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Response of yeast to oxygen

The practice of serial repitching of brewing yeast is responsible for the requirement to add oxygen to wort. Oxygen is considered to be required for synthesis of sterols and unsaturated fatty acids, both essential components of the plasma membrane (Boulton and Quain, 2001). These lipids are synthesized in the aerobic phase of fermentation and become diluted among daughter cells in subsequent growth. The quantity of oxygen supplied is one of the factors that regulate growth extent and this, in conjunction with the pitching rate and attempera- tion, are the maj or variables used to control brewery fermentations. Oxygen requirements for lipid synthesis are relatively modest, theoretically much more for sterol synthesis compared to unsaturated fatty acids, and it is likely that more is added to wort than is actually required. This prompts the question as to what is the fate of the excess?

Oxygen can function as both substrate and signalling molecule. Its presence or absence has effects at the genome level, although perhaps less than might be imagined. Based on chemostat studies under conditions of glucose-limitation, Ter Linde et al. (1999) observed that, of 6171 open reading frames, 5738 showed detectable transcripts. Of these, based on more than three times higher transcription levels, 219 showed elevated expression under aerobic conditions and 140 under anaerobic conditions. A further subset of genes are up-regulated under hypoxic conditions where oxygen is limiting (Deckert et al., 1995). The presence of these genes would be predicted in that it makes sense that cells would possess systems allowing efficient utilization of oxygen when supplies are restricted. It might be supposed that hypoxia is important for facultative anaerobes such as S. cerevisiae, since in the wild this condition would be expected to be more common than absolute anaerobiosis. Hypoxic genes include those coding for oxygen-requiring pathways, such as sterol synthesis and sterol uptake, and isoforms of aerobic enzymes, including those involved in respiration and mitochondrial ATP translocation. Two groups of genes are recognized; those with an aerobic counterpart are repressed at all but very low oxygen concentration (< 0.5 pM), and a second subset are active under all oxygen concentrations but activity is stimulated under hypoxic conditions (Becerra et al., 2002; Zitomer et al., 1997; Poyton, 1999). There are further complications since aerobic metabolism has the potential to generate harmful reactive oxygen species and the cell must have mechanisms to deal with these (Morades- Ferreira and Costa, 2000) (for further discussion of oxidative stress in yeast, see Chapter 2).

The response to oxygen requires the existence of a sensing system and the evidence suggests that haem is implicated (Kwast et al., 1999) although sterols have also been proposed (Davies and Rine, 2006), both based on the fact that oxygen is required for synthesis and therefore their intracellular concentrations would be expected to vary in relation to available oxygen levels. In the case of haem, the enzymes for the whole of the synthetic pathway are present under anaerobic conditions (Labbe-Bois and Labbe, 1990). The ROXI gene had been shown to produce a haem-induced repressor of yeast hypoxic genes, which exerts its effect by generating a product that binds to the promoter region of receptive genes (Deckert et al., 1995). An additional haem-responsive factor, Hap1, activates the expression of genes involved in respiratory functions. As oxygen levels fall, there is a concomitant reduction in levels of Rox 1 and hypoxic genes are up-regulated. Other mechanisms exist and some of these appear to be related to sterol metabolism. An element, Upc2p, has been identified (Davis and Rine, 2006) that is involved in the regulation of around a third of anaerobically expressed genes. Levels of Upc2p are linked to sterol depletion and, by implication, available oxygen.

Yeast cells respond to exogenous sterol levels in a complex fashion, which is linked to oxygen availability. Under aerobic conditions, sterols are not assimilated, a process termed aerobic sterol exclusion (Rodriguez et al., 1985). This apparently is a result of transcriptional inhibition of the sterol uptake system in which ROX1 may be implicated (Rosenfeld and Beauvoit, 2003).

The response of anaerobically grown yeast to oxygen is complex, much more so than the simple relationship between oxygen, sterols, and unsaturated fatty acids would suggest (Snoek and Steensma, 2007). The requirement for oxygen for growth can be satisfied by supplementing growth media with sterols and unsaturated fatty acids; however, it has been observed that addition of oxygen at stationary phase to such a medium increases specific fermentation rate, shortens fermentation cycle time, and increases the viability of the crop (Rosenfeld et al., 2003). The stimulation was linked to further sterol synthesis. Kwast et al. (2003) provided a description of putative roles of various classes of genes known to be induced under anaerobic conditions. Out of a total of 346 genes, 42 were related to the cell wall, 35 to cellular stress responses, 31 to carbohydrate metabolism, and 28 to the metabolism of lipids, fatty acids, and isoprenoids. In addition, several others coded for enzymes to which there was an aerobically induced isoform. In addition to the haem biosynthetic pathway, anaerobic yeast contain all the enzymes for sterol synthesis. Clearly, neither of these pathways can be active under such conditions, but it does suggest that the cell is primed in such a way that very rapid mobilization will occur should oxygen become available. This is in agreement with the observation that anaerobic yeast contains high levels of squalene, the last step in the non-oxygen requiring sterol biosynthetic pathway, and therefore the pool of this metabolite primes sterol formation should oxygen become available ( Jahnke and Klein, 1983). If this is so, it follows that lipid synthesis could proceed in cropped pitching yeast, providing oxygen is supplied. This has been shown to be the case (Boulton et al., 2000; Verbelen et al., 2009). In the work described, oxygenation of concentrated pitching yeast slurries suspended in beer was carried out, care being taken to ensure that exposure to oxygen during handling was minimized. At a temperature of 20°C, both sterols and unsaturated fatty acids were synthesized, maximum concentrations being achieved after 6-8 h. The process also resulted in loss of glycogen. In the latter study, it was shown that trehalose levels increased and the yeast acquired resistance to oxidative stress, based on the up-regulation of genes know to be implicated in these processes. Acquisition of stress resistance occurred after 45-60 min.

On a mass balance basis, the quantity of oxygen used for sterol and unsaturated fatty acid synthesis is small. The conversion of a molecule of squalene to ergosterol requires 12 molecules of oxygen. In the case of unsaturated fatty acid synthesis, desaturation of one molecule of a fatty acid requires just a single molecule of oxygen. The total quantity of sterols in yeast never exceeds around 1% of the dry weight and under brewing conditions is much less (Parks and Casey, 1995). For growth under anaerobic conditions, no more than 5 mg/l is required (Aries and Kirsop, 1978). Unsaturated fatty acids are more abundant than sterols by approximately 5-fold (Rogers et al., 1974). In addition to the requirement for oxygen, yeast cells decrease the ratio of saturated to unsaturated fatty acids as a means of maintaining membrane fluidity at lower temperature; however, not all strains have an equal facility for this (Torija et al., 2003; Beltran et al., 2008; Tronchoni et al., 2012). Although performed largely with oenological yeast strains, these studies possibly suggest that this is implicated in the ability of lager strains to grow at lower temperatures than ale types.

Under aerobic de-repressing conditions, the majority of oxygen is used by yeast as the terminal electron acceptor in oxidative phosphorylation. Under repressing conditions, yeast has an affinity for oxygen in excess of that required for sterol and unsaturated fatty acid synthesis, which prompts the question as to its role. Rosenfeld and Beauvoit (2003) reported a cyanide-resistant oxygen uptake by anaerobic yeast but concluded that this was not due to a functional respiratory chain since it could be observed in both rho+ (wild-type) and rho- (petite mutant) cells grown under anaerobic conditions. The authors suggest that synthesis of haem (2.5 molecules of oxygen per molecule), sterol, and unsaturated fatty acids account for some of this. Other oxygen-requiring pathways include the syntheses of nicotinic acid from tryptophan and ubiquinone in a pathway whose early stages are common to that used for sterol formation. The Ferri-reductase system features a plasma membrane oxidase used for high-affinity uptake of ferric ions and a mitochondrial L-proline oxidase, which is repressed under anaerobiosis but induced by proline. Interestingly, in oenological fermentations proline is rapidly metabolized in response to provision of oxygen at the end of the growth phase, presumably when the glucose and nitrogen repression signals are absent (Salmon and Barre, 1998).

 
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