Yeast Genome-Wide Expression Analysis Identifies a Strong Ergosterol and Oxidative Stress Response during the Initial Stages of an Industrial Lager Fermentation

Abstract

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.Yeast are subjected to many types of stress and metabolic challenges throughout industrial fermentation processes. These range from the physical (pressure and shearing) to the chemical, such as osmotic shock, oxidative stress, and secondary metabolite toxicity (3). Yeast fermentation is an ancient process, and years of exposure to these conditions have resulted in industrial yeast strains that have evolved mechanisms to adapt to them. However, with the advent of new processes that increase yields or are more cost-effective, different and increased demands have been placed on the yeast. This can lead to conditions that overwhelm yeast defenses, causing defective fermentations with poor sugar utilization, reduced ethanol production, and the synthesis of poor flavor qualities (21,38,55). Increased stress has also been found to have an impact on the activity and longevity properties of the yeast and therefore affect their performance in subsequent fermentations (30,47,53).To better understand the determinants of defective brews, research is often carried out by changing single parameters in model systems that can be reliably reproduced. This process has been very successful in determining the molecular mechanisms involved in protection against individual stresses. However, industrial fermentations are dynamic in nature, with multiple stresses or biological changes interacting simultaneously to cause the physiological traits of the yeast or fermentation parameters. Thus, studying the effect of individual stresses on yeast does not give the full picture of the important environmental parameters in fermentation.

Effects of Fermentation Parameters and Cell Wall Properties on Yeast Flocculation1

“…In this work, the level of zymolectins on the cell wall appears to be more or less constant during the fermentation. This finding (of constant zymolectin level) is supported by work of Higgins et al 13 and Olesen et al 25 who noted no change in FLO1 expression during fermentation of two lager strains. Interestingly, no up-regulation or down-regulation was noted of CWP1 or CWP2 genes in these studies 13,25 .…”

Carbohydrate utilization and the lager yeast transcriptome during brewery fermentation

“…Another significant outcome of this study is a clear demonstration that transcriptional responses during brewery fermentation are not necessarily comparable with the results of small-scale fermentations. Indeed, an increase in the transcription of HXT2 was observed in this study while a 50-fold downregulation was observed in another (Higgins et al, 2003a). Likewise, an increase in the transcription of many glycolytic genes was observed as the full-scale fermentation progressed, whereas a general decrease in transcription of these genes occurred during small-scale fermentation (James et al, 2003).…”

“…The high expression of HXT4, regardless of the mode of regulation, suggests that the protein encoded by this gene has an important role in hexose transport during early fermentation and that hexose transport during this period is not solely conducted by the product of HXT1, as suggested for oenological fermentations (Perez et al, 2005). While few transcription profiles for genes encoding members of this superfamily of transporters are available, Higgins et al (2003a) have reported a 50-fold downregulation in the transcription of HXT2 during a small-scale wort fermentation, which is inconsistent with the biochemical properties of the high affinity Hxt2p transporter. James et al (2003) have also reported the induction of 18 transporterencoding genes 3 days into a small-scale wort fermentation, of which seven were still highly expressed after 8 days.…”

“…The GeneChip microarray approach to monitor yeast responses during fermentation is extremely powerful in terms of the amount of transcriptional information obtained and has been successfully utilized to track the changes occurring during small-scale (James et al, 2003), pilot-scale (Higgins et al, 2003a) and production-scale (Olesen et al, 2002) wort fermentations and to identify biomarkers for zinc deficiency in wort (Higgins et al, 2003b). The disparities between the results of small-scale wort fermentations and productionscale brewery fermentations noted in this study highlight the need for more information of this kind before it may be of practical use to the brewer.…”

“…However, very few studies have utilized production strains of yeast or have involved incubation in brewery wort (Smart, 2007). Furthermore, when such studies have been performed (Higgins et al, 2003a(Higgins et al, , 2003bJames et al, 2003;Olesen et al, 2002) they have invariably involved litre-scale fermentations, which will not necessarily reflect the conditions prevalent during full-scale fermentation, which often involves wort volumes of over 5000 hl (500 000 litres). Nevertheless, such studies have greatly improved our knowledge of the transcriptome changes that occur during wort fermentation and have highlighted the complexity of this system compared to experiments in which only one environmental parameter is changed.…”