Cd 5

Fig. 3.6 Changes in levels of glycogen and sterol during the course of fermentation.

Several sterols may be found in yeasts but ergosterol is the most prominent. An excellent review of the role and diversity of sterols in yeast can be found in Parks (1978). By way of example, Behalova et al. (1994) examined the effect of growth rate on sterol synthesis in S. cerevisiae. They concluded that at low growth rates total sterol and ergosterol content of the yeast were c. 6.5% and 2-2.5%, cell dry weight, respectively.

Sterols have several putative roles in cells, both structural and functional. Sterols are important structural lipids, which play a key role in regulating membrane fluidity. Related to this, they exert other membrane effects such as regulating membrane permeability, influencing the activity of membrane-bound enzymes and affecting cell growth rate (Lees et al., 1995). The same authors describe a 'hormonal' role of specific yeast sterols, at low concentration (10 nM), in which they are vital to progression through the cell cycle. In addition to the role in membrane structural function, Parks et al. (1995) implicated sterols in amino acid and pyrimidine transport, in resistance to anti-fungal agents and some cations, as well as being required for the development of respiratory function.

With respect to membranes, sterols are considered to fulfil a vital role in maintaining membrane fluidity. Presence of sterols in membranes are reported to fulfil two

Fig. 3.6 Changes in levels of glycogen and sterol during the course of fermentation.

roles, described as 'sparking' and 'bulk' functions (Rodriguez & Parks, 1983; Lorenz et al., 1989). These authors suggested that, provided small 'sparking' quantities of ergosterol were present in the membrane, then other sterols could substitute for ergosterol and fulfil a bulk role. The critical feature of the sparking sterol was the presence of the 24 (3-methyl group of ergosterol and it was essential that at least a small quantity of this lipid was present in membranes in order to maintain proper fluidity.

Rodriguez et al. (1985) recognised four groups of sterols - described as sparking, critical domain, domain and bulk, based on work with a sterol auxotroph of S. cerevisiae termed 'RD-5'. Ergosterol could fulfil all of these roles. The minimum concentration of free sterol below which cell proliferation could not occur was defined as the domain function. No growth was possible unless a small quantity of ergosterol was present, in combination with other sterols, and this was ascribed to the requirement for the 24(3-methyl group. In the presence of ergosterol the bulk membrane function could be fulfilled by cholestanol. The mutant strain could grow in the presence of lanosterol (5|igml *) and ergosterol (lOOngml *) but not lanosterol alone. The additional lOOngml 1 ergosterol was insufficient to control overall membrane fluidity alone and it was concluded that this was needed in very restricted areas of the membrane, termed the 'critical domain function'.

Yeast cells accumulate sterols into membranes until the bulk requirement is satisfied. Further synthesis may continue, although in this case the additional sterol is esterified to long-chain fatty acids and deposited in intracellular lipid vesicles (see Section 4.1.2.5). The most abundant sterol component of the steryl ester pool is zymosterol (Leber et al., 1992). In aerobic batch cultures, steryl esters accumulate during the stationary phase of growth. When such cells are re-inoculated into fresh medium rapid hydrolysis of steryl esters occurs and the liberated sterol incorporated into growing membranes (Quain & Haslam, 1979; Lorenz & Parks, 1991). In this sense, the steryl esters may be viewed as 'a store of sterols'.

Lewis et al. (1987) concluded that yeast cells must maintain a low level of free sterol, which is essential for growth. Provided there is sufficient sterol available, a further expandable free sterol pool may be maintained. However, as this pool of free sterol increased in size this was accompanied by a progressively increased rate of sterol esterification. The extent of esterification could be correlated with activity of acyl-CoA ergosterol acyltransferase.

The importance of steryl ester accumulation in brewing yeast strains during fermentation is not known. Thus, significant esterified sterol accumulation is associated with derepressed aerobic growth. This pool accounts for the five-fold difference in total sterol concentration of repressed and de-repressed cells (Quain & Haslam, 1979). In this regard it might be predicted that esterification would be of relatively small significance in brewing yeast. However, investigation of the biochemistry underlying the differences between high and low oxygen requiring brewing yeast strains has revealed that sterol esterification could be implicated. Thus, a comparison of sterol spectra revealed that the high-oxygen-requiring strain contained a relatively high concentration of zymosterol. This sterol is not readily incorporated into membranes and is usually esterified. Therefore, this implies that the high oxygen requiring strain would expend more oxygen to produce the same quantity of free membrane sterol as the low-oxygen-requiring strain (S.C.P. Durnin, personal communication).

In addition, when pitching yeast is deliberately exposed to oxygen under non-growing conditions some limited sterol synthesis may occur. This phenomenon has been used in a process designed to improve fermentation performance consistency (see Section 6.4.2.2). Here it was observed that during oxygenation of freshly cropped pitching yeast the total intracellular sterol content increased approximately five-fold from 0.2% to 1% of the total cell dry weight. In other words, a similar increase in total sterol concentration as is seen in a conventional oxygenated wort fermentation. However, analysis of the sterol spectrum revealed that ergosterol and zymosterol were synthesised in roughly equal proportions, and, furthermore, as a proportion of the whole, zymosterol increased at the expense of ergosterol (see Fig. 6.24). How the pool of steryl ester so formed would then influence subsequent fermentations remains unclear.

Sterols can be assimilated by yeast from the medium, however regulation is complex. Salerno and Parks (1983) reported that exogenous sterol could only be accumulated by anaerobic cells or mutant strains that are auxothrophic for sterol. Conversely, aerobic yeast capable of sterol synthesis did not utilise exogenous sterol. Parks' laboratory christened this phenomenon 'aerobic sterol exclusion' and has demonstrated that the process is associated with haem metabolism. Thus, several of the enzymes involved in the sterol biosynthetic pathway are haemoproteins. Not surprisingly therefore, haem deficient mutants are auxotrophic for sterol. Haem sufficiency, which requires aerobiosis, apparently derepresses sterologenesis and simultaneously inhibits sterol uptake (Lorenz & Parks, 1987; Shinabarger et al., 1989). The precise mechanisms for this phenomenon remain to be fully elucidated; however, it would suggest that during a brewery fermentation, sterol uptake could only occur after the initial aerobic phase was over.

Sterols are synthesised using carbon devolving from glycolysis via acetyl-CoA as part of the general pathway leading to the formation of branched isoprenoids. The first part of the synthesis is an anaerobic process, which involves the conversion of acetyl-CoA to squalene. An intermediate of this pathway is farnesyl pyrophosphate, which forms a branch-point between sterol synthesis and the formation of haem and ubiquinone, both essential components of the electron transport chain (Fig. 3.7).

In the terminal part of the sterol biosynthetic pathway devolving from squalene, molecular oxygen is used to form 2,3-epoxysqualene, followed by cyclisation to form the first sterol, lanosterol. Other sterols, including ergosterol are formed from lanosterol in a complex pathway in which the precise reactions vary from one yeast strain to another (Fig. 3.8). Apart from the initial epoxidation of squalene, molecular oxygen is involved in desaturation reactions involving cytochrome P450 mono-oxygenases.

Predictably, the regulation of sterol synthesis in yeast is complex! Of particular note is the influence of intracellular compartmentalisation (see Section 4.1.3). The sites of sterol synthesis and ultimate deposition may be distinct and therefore, regulation of synthesis and intracellular transport must be co-ordinated processes. Free sterols may be found in greatest abundance in the plasma membrane but smaller quantities are also found in the membranes surrounding other intracellular organelles such as the

Acetoacetyl-CoA -►

Hydroxymethylglutaryl-CoA 2NADPH--► Coenzyme A

Mevalonic acid

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