Utp

UDP-glucose

Glycogen synthase

Branching enyme

Glycogen

Fig. 3.4 Glycogen biosynthetic pathway.

sugar. For example, sulphur, nitrogen and phosphorus limitation (Lillie & Pringle, 1980). Glycogen accumulation may also occur under carbon limitation, for example, during diauxic growth on glucose. In this case when glucose falls to a concentration below that required to saturate the uptake system growth rate is restricted and glycogen accumulation is triggered. Accumulation continues until the exogenous sugar becomes exhausted. This glycogen store provides an energy source for induction of the respiratory and gluconeogenic systems, which are required for utilisation of ethanol (Gancedo & Serrano, 1989).

Regulation of glycogen synthase and phosphorylases activities is complex, as would be predicted from the number of external signals which can influence glycogen levels in yeast. Simultaneous accumulation and utilisation are possible, overall flux being controlled by co-ordinate regulation of glycogen synthase and glycogen phosphorylase. Wills (1990) has reviewed some of the mechanisms involved. Glycogen phosphorylase is activated by phosphorylation and deactivated by dephos-phorylation. Glycogen synthase is activated by dephosphorylation. Two active forms of the enzyme occur, one of which is inhibited by glucose 6-phosphate. Apart from covalent modification to the enzymes, activity is also modulated by intracellular adenine nucleotide concentrations. Phosphorylation and dephosphorylation of the glycogen synthases and phosphorylase is regulated by signal transduction cascade pathways involving cyclic AMP and non-cyclic dependent kinases.

In the context of brewery fermentation it has been suggested that glycogen fulfils two vital roles. First, it provides the carbon and energy for synthesis of sterols and unsaturated fatty acids during the aerobic phase of fermentation and, second, energy for cellular maintenance functions during the stationary phase of fermentation and in the storage phase between cropping and re-pitching (Quain & Tubb, 1982). When yeast is pitched into aerobic wort there is an immediate mobilisation of glycogen reserves and this is accompanied by synthesis of sterol (Fig. 3.6). The period of rapid glycogen dissimilation is terminated by the disappearance of oxygen from the wort. The subsequent phase of rapid fermentation and yeast growth is associated with glycogen accumulation. Maximum glycogen concentrations are reached towards the end of primary fermentation after the point where yeast growth has ceased. In the final stationary phase when primary fermentation is complete, glycogen levels decline slowly.

It may be surmised that oxygen is the primary trigger for glycogen dissimilation immediately after pitching and that this is necessary to provide energy and carbon for sterol synthesis since exogenous sugars cannot be utilised due to lack of membrane function in the sterol-depleted pitching yeast. During the latter part of active primary fermentation, glycogen assimilation is favoured due to the presence in wort of the high ratio of sugars to other components. Exhaustion of nutrients other than sugars will gradually reduce yeast growth rate and progressively favour greater glycogen accumulation as primary fermentation progresses. When fermentable sugars become exhausted from the wort and anaerobiosis precludes utilisation of ethanol, glycogen dissimilation will be favoured in order to allow the yeast to withstand the starvation conditions.

The apparent importance of glycogen in fermentation has implications for storage of pitching yeast. Since glycogen is utilised for maintenance functions during storage it is important to remove yeast crops from fermenter as soon as is practicable in order to prevent excessive glycogen degradation in the period between the end of primary fermentation and cropping. Cooling of fermenter contents is useful in this respect. In storage vessels low temperatures also reduce yeast metabolic activity and conserve glycogen stores. Even so, the need to have sufficient glycogen to fuel sterol synthesis during subsequent fermentation serves to limit the time for which pitching yeast can be safely stored without compromising subsequent fermentation performance. In practice, this is usually no longer than 3 days at 2^°C, under an inert gas atmosphere. For an exhaustive review of the impact of yeast storage conditions on glycogen turnover see Section 7.3.

The observation that rapid glycogen dissimilation may be triggered in anaerobic yeast suddenly exposed to oxygen implies that this may occur in stored pitching yeast exposed to air. In this case limited sterol synthesis may ensue and failure to correct for this by reducing wort oxygen concentrations will result in excessive yeast growth and loss of fermentation efficiency on re-pitching. For this reason yeast should be stored under an atmosphere of nitrogen or carbon dioxide. Alternatively, the ability of yeast to couple glycogen dissimilation to sterol synthesis in a controlled process which has the potential to remove the need for wort oxygenation has been proposed (Boulton et al., 1991; Masschelein et al., 1995). This is described in detail in Section 6.4.2.2.

3.4.2.2 Trehalose. Trehalose is a disaccharide (a-D-glucopyranosyl-l,l-a-D-glucopyranoside) which contains two molecules of D- glucose. Like glycogen it is also synthesised in reactions which utilise uridine diphosphate as a carrier of glucose molecules (Fig. 3.5). The key enzyme is trehalose phosphate synthase (UDPG-glucose 6-phosphate transglucosylase) which catalyses the transfer of a glucose residue from uridine diphosphate glucose to glucose 6-phosphate. A phosphatase liberates the phosphate group forming trehalose. Synthesis of trehalose may derive from glucose or from glucosyl residues derived from degradation of glycogen. Furthermore, it is suggested that trehalose phosphate synthase has a greater affinity for UDP glucose than does glycogen synthase (Gancedo and Serrano, 1989). Mobilisation of trehalose occurs using another enzyme, trehalase, of which two occur in yeast, one which is associated with vacuoles and another which is cytosolic (Panek & Panek, 1990).

Glucose

Fig. 3.5 Trehalose biosynthetic pathway.

It has been suggested that trehalose 6-phosphate may have a regulatory role in glycolysis in yeast by feedback control of hexokinases (Blâzquez et al., 1993). This perhaps suggests that in some circumstances it may act as a potential energy reserve. Both trehalose phosphate synthase and trehalase are subject to complex regulation in a similar manner to glycogen. Thus, cyclic AMP-dependent phosphorylation and dephosphorylation reaction are responsible for activation and deactivation of the enzymes leading to trehalose accumulation and degradation.

Like glycogen, trehalose also accumulates in yeast under conditions of nutrient limitation and therefore it was logical to conclude that it also served as a storage carbohydrate (Lillie & Pringle, 1980). However, the apparent redundancy of having two storage systems together with the element of interconvertability is perhaps indicative of differing roles for each polysaccharide. In this respect the same authors observed that although trehalose accumulation occurred under conditions of nutrient limitation this took place after glycogen formation and indeed there was evidence that glycogen was mobilised to provide glucose residues for trehalose formation. This was taken to indicate that trehalose did not behave like a typical storage polysaccharide. The low observed trehalose concentration in yeast during exponential growth is a consequence of glucose repression and inactivation. Thus, under repressed conditions the presence of glucose results in elevated levels of cyclic AMP due to activation of adenylate cyclase. This in turn activates protein kinase phosphorylase and thence activation of trehalase.

Simultaneously, a cyclic AMP-dependent phosphorylase converts the trehalose 6-phosphate synthase into an inactive phosphorylated form. In derepressed cells, such as is the case in the stationary phase of the growth cycle, these effects are reversed and trehalose accumulation is favoured. Glycogen synthase is regulated in a similar manner to trehalose synthase; however, the repressing effects of glucose are quantitatively greater in the case of trehalose synthetic pathway (Van der Plaat & van Solingen, 1974; Entian & Zimmermann, 1982; Uno et al., 1983; Londesborough & Varimo, 1984; Thevelein, 1984; Mittenbuhler & Holzer, 1988; Panek & Panek, 1990; Winkler et al., 1991). This - in combination with the already made observation that trehalose synthase has a greater affinity for UDP-glucose compared to glycogen synthase - provides an explanation for the observation that in the stationary phase of growth accumulation of glycogen occurs before that of trehalose.

Trehalose is known to confer resistance to heat and desiccation in a diverse range of organisms such as insects, plants, yeast and higher fungi. In addition, it is associated with spore formation (Elbien 1974; Crowe et al., 1984; Neves & Francois, 1992; de Virgilio et al., 1994). Colaco et al., (1992) described the ability of trehalose to stabilise protein structure such that in the presence of this disaccharide many enzymes exhibit startling resistance to heat and desiccation. Iwahashi et al. (1995) used whole cell NMR analysis of yeast and concluded that trehalose protected cells from temperature extremes by stabilising membrane structure. Further circumstantial evidence for a role for trehalose distinct from that of simple storage carbohydrate is provided by the fact that its synthesis requires metabolic energy, whereas no ATP is generated in its dissimilation. Trehalose has been shown to be a most effective agent for preventing damage to membranes by its ability to prevent phase transition events in lipid bilayers. The mechanism of action appears to be via binding of the hydroxyl groups of the sugar to the polar head groups of phospholipids in locations otherwise occupied by water. For maximum effectiveness, trehalose requires to be present at both the inner and outer surfaces of the membrane (Crowe et al., 1984).

It follows that in order for trehalose to exert its protective effects it must be transported from the site of synthesis in the cytosol to the membrane. Kotyk and Michaljanicova (1979) demonstrated the presence in S. cerevisiae of a transporter which rendered the cells capable of taking up exogenous trehalose. Eleutherio et al. (1993) postulated that the same carrier was responsible for transporting intracellular trehalose to the periplasm and inner membrane. The presence of this carrier was essential for the protective effects of trehalose to be expressed. Thus, mutant strains with no carrier could not withstand dehydration although trehalose accumulation was unimpaired. The same mutants could be afforded protection from dehydration by the addition of exogenous trehalose. As with trehalose accumu lation, the activity of the carrier was repressed by glucose and was only active in stationary phase cells.

Yeast, in common with other eukaryotes, exhibits a heat shock response. This phenomenon is triggered when cells are exposed for a short period to a high but non-lethal temperature at which growth is not permitted. Such cells returned to a growth-permitting temperature exhibit an increased but transient tolerance to subsequent exposure to lethal temperatures. The effect is associated with the synthesis of a large number of so-called 'heat shock proteins' (Lindquist & Craig, 1988; Schlesinger, 1990). Acquisition of thermotolerance by expression of the heat shock response provides protection against other stresses. For example, increased tolerance to high osmotic pressures and ethanol (Piper, 1995). Indeed exposure to ethanol above a threshold concentration induces a similar response to heat shock (Piper et al., 1994).

There is much evidence to suggest that increased tolerance to stress is associated with accumulation of trehalose. For example, Sharma (1997) reported that in a strain of S. cerevisiae increased osmotic pressure brought about by elevated levels of sodium chloride was accompanied by increased accumulation of trehalose and increased tolerance to ethanol. A similar correlation between trehalose content and stress resistance in yeast was reported by Attfield et al. (1992). Elliott et al. (1996) isolated a mutant yeast strain which was unable to acquire heat shock resistance. The strain did not accumulate trehalose. The evidence suggests that induction of heat shock proteins and trehalose accumulation are related (Hottiger et al., 1989; Fujita et al., 1995).

The observation that trehalose accumulation occurs during the late stationary phase can be explained in that it represents a physiological response to cells undergoing transition. Thus, in resting cells glycogen provides a readily utilisable source of carbon for maintenance energy. A proportion of this carbon, supplemented with exogenous sugar, is utilised for trehalose synthesis. The trehalose pool provides protection for cells during the resting phase. A variation of the transition from growth to resting state is differentiation into spore formation. In this case trehalose is the major carbon store and is essential for germination.

Rapid accumulation of trehalose in response to environmental changes may be taken as evidence that trehalose synthase acts as a stress protein. The mechanism of this adaptive response is believed to involve a general stress responsive promoter element (STRE) which has been identified in the upstream regions of several yeast genes. These include the TPS2 gene, which codes for a subunit of the trehalose phosphate synthase complex. The element is derepressed by nitrogen starvation in stationary phase cells and by applied stresses (Mansure et al., 1997).

The role of trehalose in brewing fermentation is unclear. It is accepted that high trehalose levels are important in commercial preparations of bakers' yeast, particularly active dried yeast (Trivedi & Jacobsen, 1986). In this case, trehalose provides protection during the drying phase. It also plays a role in providing cryoprotection when the yeast is used in frozen doughs. Bakers' yeast fermentations are conducted in such a way that trehalose is maintained at high levels, typically 15-20% of the dry weight, with 10% being considered critical (Gelinas et al., 1989). In brewing yeast cropped from fermenter trehalose concentrations tend to be modest, usually less than 5% of the dry weight. Higher levels may accumulate in yeast cropped from very-high-gravity fermentations.

Majara et al. (1996a and b) investigated the accumulation of trehalose in lager yeast fermentations using worts of varying gravities between 11 and 25°Plato. The higher-gravity worts were obtained by supplementation of low-gravity worts with glucose. It was demonstrated that trehalose accumulation occurred in late fermentation at a concentration proportional to the gravity of the wort. Thus, trehalose accounted for no more than 2-3% of the yeast dry weight in an 11 "Plato wort, whereas this increased to 20-25% of the cell dry weight in yeast removed from a 25°Plato wort. The effect was independent of the ethanol concentration; however trehalose accumulation was favoured in the presence of sorbitol and could be correlated with the formation of glycerol. For these reasons the authors concluded that trehalose accumulated in response to osmotic stress. Other applied stresses such as freezing, exposure to ethanol, oxygenation, drying and carbonation all resulted in trehalose accumulation. It was concluded, therefore, that the detection of unexpectedly high concentrations of trehalose in pitching yeast should be taken as a sign that the yeast had been subject to stress.

It was contended that trehalose may be relatively unimportant as a stress protectant during early primary fermentation, as the presence of glucose would tend to mitigate against its synthesis and even when formed the repression of the carrier would not allow transport to the membrane for trehalose to exert a protective effect. In this regard, where very-high-gravity fermentation is practised it would be sensible to avoid high concentrations of glucose to promote early trehalose synthesis and transport. However, trehalose is important during storage of pitching yeast since these cells are derepressed and therefore the carrier is fully active. In this case, the conditions should be controlled to minimise trehalose degradation. In other words, short storage times, low temperatures and the absence of oxygen.

The optimum concentration of trehalose required by brewing yeast for it to exhibit resistance to applied stresses remains to be identified. Presumably, when sufficient is available on each side of the membrane for the stabilisation effect to occur no additional pool is required unless there is a continual dynamic turnover of synthesis and degradation. It would be interesting to know what this minimum required concentration is. In brewing fermentations, as discussed already, trehalose accumulation can be correlated with starting gravity, an effect ascribed to the increasing osmotic stress associated with elevated solute levels. It could also be argued that the increased accumulation of trehalose simply reflects the greater availability of carbon (or high carbon to nitrogen ratio) in very-high-gravity fermentations. After all, under the conditions of production brewing even a 10°Plato wort must present a considerable stress to yeast. However, under these conditions trehalose typically accounts for only 2^1% of the yeast dry weight even when the repressing effects of glucose are alleviated in late fermentation. During a study of sugar assimilation from a 300 hi 10°Plato ale fermentation, it was observed that a transient peak occurred when growth had ceased, which was tentatively identified as trehalose (C.A. Boulton, unpublished data). Presumably, loss of trehalose to the medium could be of more general occurrence, and overall biosynthetic rates may be greater than assumed, even in worts of moderate concentration.

An alternative to the usual practice of supplying wort with oxygen at the start of fermentation, is direct oxygenation of pitching yeast (see Section 6.4.2.2). It may be demonstrated that this process promotes sterol synthesis at the expense of glycogen dissimilation (Boulton et al., 1991). Callaerts et al., (1993) reported that the oxygenation process also resulted in trehalose accumulation in a pattern which showed a positive correlation with sterol formation. Presumably this was also a response to stress, in this case due to oxygen. The carbon for trehalose accumulation must have been provided by glycogen dissimilation.

3.4.3 Fermentable growth medium induced pathway

The phenomenon of glucose catabolite repression does not require growth for expression. Many of the metabolic events associated with yeast growth on wort are clearly growth related. For example, regulation of carbon flow between glycolysis and accumulation of polysaccharide reserves such as glycogen and trehalose (Section 3.4.2). Thevelein & Hohmann (1995) have developed a concept which they have termed the 'fermentable growth medium induced pathway' (FGMIP). This is defined as one or more signal transduction pathways, which are activated by a specific combination of nutrients, readily fermentable sugars and all other nutrients required for growth. The authors consider that this is distinct from the general glucose repression and RAS-adenylate cyclase pathways.

The characteristics and a summary for the evidence of the existence of this pathway is provided in Thevelein (1994). When yeast is starved for an essential nutrient such as nitrogen, sulphate or phosphorus in the presence of glucose the cells arrest in the G1 phase of the cell cycle and then enter the resting phase, G0 (see Section 4.3.3.1). This is associated with accumulation of glycogen and trehalose, induction of heat shock proteins and repression of ribosomal protein genes. Subsequent addition of the missing nutrient induces growth and a rapid loss of stationary phase characteristics. Thus, there are rapid post-translationally induced changes in enzyme activity and gene expression which reverse the changes described above. Similar changes may be observed when exponentially growing fermentative cells are made to undergo a sudden increase in growth rate. This does not happen with cells growing on non-fermentable carbon sources.

Cells arrested in G0 phase by starvation for a nutrient other than sugar are triggered to activate phosphofructokinase, glycogen synthase, glycogen phosphorylase and inactivate trehalose 6-phosphate synthase and trehalose 6-phosphate phosphatase by the addition of the missing nutrient. However, this occurs only in the presence of glucose. Inhibition of protein synthesis does not prevent the occurrence of these events. Addition of the missing nutrient does not produce a sudden spike in cyclic AMP, which was taken to indicate that this metabolite was not acting as a secondary messenger. Instead it is argued that FGMIP in some way activates free catalytic subunits of cyclic AMP-dependent protein kinases.

Individual pathways are still to some extent overlapping. Thus, cells starved for nitrogen in the presence of glucose are glucose repressed. Addition of nitrogen induces FGMIP but the cells remain repressed. Presumably this is similar to the effects of oxygen in brewing fermentation being modified by the presence of repressing concentrations of sugar. The continued effects of repression were taken to indicate that expression of FGMIP did not require glucose-induced activation of the

Ras adenylate cyclase pathway since the latter is itself glucose repressible. Confirmation was provided by the observation that activation of FGMIP does not require glucose phosphorylation whereas this is mandatory in the RAS adenylate cyclase pathway. However, it was tentatively concluded that the initial glucose sensing mechanism for the RAS and FGMIP systems could be the same, although the different requirements for phosphorylation cast doubt on this premise. The search for the glucose sensor continues.

The reverse of a fermentation growth medium induced pathway is that which might come into play in stationary phase under starvation conditions. This has been termed a stress-induced (STRE) pathway and is described as a stationary phase co-regulated gene induction which is a response to starvation stress and is distinct from the heat shock response. Thevelein (1994) has also discussed this possibility within the context of FGMIP. The STRE pathway hypothesis states that under conditions of growth certain genes are not expressed. It has been reported that exhaustion of glucose, transfer to a non-fermentable carbon source or starvation of an essential nutrient results in the expression of the STRE genes. Thus, this is a general stress-sensing pathway and nutrient limitation would only be one of many triggers of the induction of it. In this sense a mechanism must exist for sensing sub-optimal concentrations of pertinent substrates.

It was argued that the existence of such a general stress-sensing pathway which involved both nutrient limitation and other stresses was unproved. Instead different stimuli may produce similar phenotypic effects by the use of the same target sites. For example, FGMIP and RAS adenylate cyclase pathway both interacting with cyclic AMP-dependent protein kinase. As the author points out, new signal transduction systems in yeast are being regularly discovered. Further elucidation of the structure and function of the yeast genome together with the advent of proteomics (see Section 4.3.2) will no doubt bring further clarification to this complex but fascinating aspect of yeast metabolism.

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