SEARCH

SEARCH BY CITATION

Keywords:

  • ethanol sensitivity;
  • cell integrity;
  • Calcofluor white;
  • high-gravity fermentation;
  • GFP

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

The Saccharomyces cerevisiae deletion collection was screened for impaired growth on glucose-based complex medium containing 6% ethanol. Forty-six mutants were found. Genes encoding proteins involved in vacuolar function, the cell integrity pathway, mitochondrial function, subunits of the co-chaperone complex GimC and components of the SAGA transcription factor complex were in this way found to be important for the growth of wild-type Saccharomyces yeast in the presence of ethanol. Several mutants were also sensitive to Calcofluor white (14 mutants), sorbic acid (9), increased temperature (5) and NaCl (3). The transcription factors Msn2p and Ars1p, tagged with green fluorescent protein, were translocated to the nucleus upon ethanol stress. Only one of the genes that contain STRE elements in the promoter was important under ethanol stress; this was TPS1, encoding trehalose 6-phosphate synthase. The map kinase of the cell integrity pathway, Slt2p, was phosphorylated when cells were treated with 6% ethanol. Two out of three mutants tested fermented 20% glucose more slowly than the wild-type. Copyright © 2006 John Wiley & Sons, Ltd.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

In the process of beer fermentation, the primary role of yeast is the conversion of wort sugars to alcohol and carbon dioxide. During fermentation, yeast is exposed to many environmental stresses, to which it responds by adjusting its gene expression (Olesen et al., 2002; James et al., 2003). These stresses become more severe when high-gravity worts, containing high concentrations of fermentable sugars, are used. Initially the yeast will be exposed to high osmolarity caused by the high sugar concentrations. In the fermentor the yeast has to face problems such as anaerobiosis, high ethanol concentrations, internal acidification and depletion of nutrients. All these factors affect vitality and viability of the yeast (Gasch and Werner-Washburne, 2002). Knowledge of how the yeast adapts to these conditions will be useful for the selection of improved strains for high-gravity applications.

Many details are known about the stress responses of yeast. Sensing osmotic stress involves the plasma membrane proteins Sln1p and Sho1p, which activate a signal transduction pathway, leading to the activation of the MAP kinase Hog1p. Hog1p activates at least five transcription factors, Hot1p, Sko1p, Smp1p, Msn2p and Msn4p, leading to the expression of genes involved in glycerol synthesis and osmostress resistance (Hohmann, 2002). The latter two transcription factors are also involved in the general stress response. One of the genes most responsive to high osmolarity is the yeast glycerol transporter Stl1p (Ferreira et al., 2005). Although the Hog1p pathway is activated, osmotic stress was suggested not to be critical for high-gravity fermentation (Hammond, 2001).

The presence of low-molecular-weight organic acids in combination with a low pH leads to diffusion of the protonated acids through the plasma membrane of the yeast cell. The neutral pH of the cytosol will result in dissociation of the acids and thus often in internal acidification. The ABC transporter Pdr12p is involved in extrusion of these dissociated acids and is essential for organic acid tolerance (Piper et al., 1998). In energized cells, the plasma membrane ATPase Pma1p maintains the internal pH by pumping out protons (Holyoak et al., 1996). However, this results in a futile cycle, as the acids will be protonated again in the growth medium. Yeast adapts its gene expression to the presence of acids in the medium (de Nobel et al., 2001). Mutations resulting in hyperresistance or hypersensitivity to sorbic acid have been identified recently (Mollapour et al., 2004).

Saccharomyces yeasts have also developed an array of stress responses and adaptation mechanisms towards ethanol. These include the upregulation of heat shock proteins, stimulation of the plasma membrane ATPase and regulation of plasma membrane lipid composition (Piper, 1995; Swan and Watson, 1997; Fernandes and Sa-Correia, 2003). Microarray analysis revealed a number of genes regulated during ethanol stress (Alexandre et al., 2001). Some genes required under ethanol stress were identified by transposon mutagenesis (Takahashi et al., 2001). A number of these genes may be regulated by the general stress transcription factors Msn2p and Msn4p. However, recently a transcription factor responding specifically to ethanol stress was identified (Betz et al., 2004). We exploited the availability of the complete collection of yeast deletion mutants to screen for genes essential under ethanol stress.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

Strains and media

S. cerevisiae strains used in this study comprise the genetic background of BY4742 (Brachmann et al., 1998). The collection of single deletion mutants in BY4742 was obtained from EUROSCARF. Batch cultures of yeast were grown aerobically in complex medium (YP, yeast extract 1% w/v, peptone 2% w/v) supplemented with 2% w/v glucose (YPD). The plates also contained 2% agar. Media were supplemented with ethanol (6% v/v), Calcofluor white (50 µg/ml), NaCl (0.4 M) or sorbic acid (0.5 mM). Medium with sorbic acid was adjusted to pH 4.5 with hydrochloric acid. Fermentations were carried out in YP supplemented with 20% glucose. Unless otherwise indicated, strains were grown at 30 °C. The initial mutant screen was carried out by replica-plating the complete EUROSCARF collection onto YPD medium. After growth, colonies were lifted with a 96-pin tool and diluted in 96-well plates containing 100 µl sterile water. The resulting cell suspensions were plated onto 7% ethanol plates using the same 96-pin tool. The plates were incubated in a lidded container to minimize evaporation of ethanol from the plates. A beaker with 7% ethanol was included in the same container. The plates were photographed after 2 and 5 days of growth. Drop tests were performed from cell suspensions containing approximately 1 × 106 cells/ml. Ten-fold serial dilutions were made and 5 µl each suspension was applied onto a diagnostic plate. Ethanol-containing plates were incubated as described above. The plates were photographed after 2 and 5 days.

GFP fusion proteins

For expression of GFP fusion proteins, we made use of pUG35 and pUG36 (Güldener and Hegemann, unpublished), centromere-based vectors containing the MET25 promoter, a polylinker and a sequence encoding yeast-enhanced GFP. Fusions can be made at the carboxy- (pUG35) or amino-terminus of a protein (pUG36). pUG35-MSN2 was constructed by amplifying MSN2 from the genome with a proofreading polymerase (Dynazyme, Finnzymes, F505L), using primers 5′-GGC CGG ATC CAT GAC GGT CGA CCA TGA TTT C-3′ and GGC CAA GCT TAA TGT CTC CAT GTT TTT TAT GAG-3′. The amplified fragment was inserted into pUG35 using the restriction sites BamHI and HindIII. pUG36-ASR1 was constructed by amplifying ASR1 from the genome with the proofreading polymerase, using primers 5′-GCC GAT ACT AGT ATG GAA GAG TGT CCT ATT TG-3′ and 5′-CGG CGA AAG CTT CTC ATC ATG ACA GTA AAT TAG-3′. The amplified fragment was inserted into pUG36, using restriction sites SpeI and HindIII. Strains were transformed with pUG35-MSN2 or pUG36-ASR1. Yeast transformations were performed by the lithium acetate method (Gietz and Woods, 2002). The cells were grown overnight in SC medium without uracil. Then cells were diluted until OD600 ≈ 0.25 in the same medium and allowed to reach OD600 = 0.6. Subsequently the cells were shifted to SC medium without uracil and methionine to induce the MET25 promoter. After 4–5 h of induction the culture was visualized by fluorescence microscopy. The remaining culture was then shifted to medium containing 6% ethanol in order to activate the stress pathways. Cells were visualized during 1 h of growth in glucose or in the presence of ethanol. A Leica TCS SP2 confocal scanning microscope was used to obtain the pictures with an excitation wavelength for GFP at 488 nm and selection of emitted light of 500–570 nm.

Western blot

Overnight cultures were diluted to OD600 = 0.2 and grown to OD600 = 1. Subsequently the cultures were incubated in the indicated media for 1 h. Then two OD600 units were harvested by centrifugation and immediately lysed by addition of 100 µl SDS-sample buffer preheated at 95 °C and further incubation at 95 °C for 10 min. 10 µl each sample was applied to a 4–12% Polyacrylamide Bis-Tris SDS gel (Invitrogen) and separated according to the manufacturer's instructions. Protein was transferred to nitrocellulose filters using semi-dry electroblotting (Kyhse-Andersen, 1994). The filters were incubated with polyclonal antibody against Slt2p (Santa Cruz, sc-20168) or phospho-p44/p42 MAP kinase (Cell Signalling, #9101s) in 20 mM Tris–HCL, pH 7.5, 140 mM NaCl (TBS) containing 2% BSA. The filters were washed briefly with TBS + 2% BSA and incubated with horseradish peroxidase conjugated rabbit IgG (Dako Cytomation), washed again once with TBS containing 2% Tween 20 and once with TBS. Reacting polypeptides were visualized using the ECL Plus Western Blotting Detection System (Amersham Biosciences) and a Storm 860 Scanner (Molecular Dynamics).

Small-scale high-gravity fermentations

For stirred mini-aerobic fermentations, 3 ml pre-culture grown for 72 h in YPD was used to inoculate 200 ml YP medium supplemented with 20% glucose to OD600 = 0.2. Cultures were incubated at 25 °C in 200 ml measuring cylinders continuously stirred at 130 r.p.m. and covered with an inverted beaker. The weight of each cylinder was determined at the start and subsequently at all indicated time points. Weight loss by evaporation was corrected for by following the weight of a parallel cylinder without added yeast. The density of the medium in degrees Plato was determined using a densitometer (Density Meter, Anton Paar DMA35n) at the beginning and end of the fermentation. The Plato scale is routinely used in the brewing industry. One degree Plato represents a sugar content equivalent to 1% sucrose by weight (de Clerck, 1957). The weight loss at all intermediate points was used to calculate the density of the culture medium at those time points.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

Identification of ethanol-sensitive mutants

To identify ethanol-sensitive mutants, we replica-plated the complete EUROSCARF collection, consisting of 4868 deletion mutants, onto glucose-based medium containing 7% ethanol. Initially, 526 strains displayed apparently reduced growth. Estimating the difference between growth and slow growth is difficult to do by eye on plate media. Therefore, the 526 strains were first tested by re-streaking on plates containing 6% ethanol. This resulted in approximately 200 strains with reduced growth that were further tested by plating serial dilutions on 6% ethanol-containing plates. Strains that displayed a clear reduced growth phenotype were in this way unambiguously identified. These tests finally identified 14 strains that did not grow and 20 strains that showed a clear slow-growth phenotype on 6% ethanol. Independent isolates of both mating types were re-tested and showed identical phenotypes. The remaining deletion strains displayed no or only a weak growth phenotype on plates containing 6% ethanol. Figure 1 shows an example of some of the phenotypes. The 34 mutations were grouped according to gene annotation. In each group other genes with similar functions were tested on ethanol plates. This identified 12 further mutations resulting in slow growth on ethanol-containing medium. The 46 genes identified are shown in Table 1. Bold characters indicate the genes identified in the initial screen.

thumbnail image

Figure 1. Growth phenotypes of the wild-type and representative mutants on ethanol-containing medium (6% v/v). Ten-fold serial dilutions were spotted onto YPD plates without (Y) and with (E) ethanol. Plates were incubated at 30 °C for 5 days

Download figure to PowerPoint

Table 1. List of genes whose deletion leads to growth deficiency on complex glucose-based medium containing 6% ethanol
 Mitochondrial functionVacuolar functionPkc1 pathwayActin/tubulin foldingHistone acetylationMiscellaneous
  1. The genes are listed in functional categories. The genes identified in the initial screen are shown in bold.

No growthIMG1VMA10BEM2 KAR3
VPH1SMI1 RIB4
VPS15 RMD8
VPS16 SEC66
VPS34 
VPS36 
VPS39 
Reduced growthATP1FAB1ROM2GIM4ADA3FEN1
HMI1VAC14SIT4GIM5GCN5ERG6
MSK1VPS5SLG1 SPT3PAT1
MTF2VPS9SLT2 SPT7PLC1
 VPS24SWI4  POP2
VPS29 RAD6
VPS30 SIN3
 SUR4
TPS1
YDL114W

Five mutations are involved in mitochondrial function and only one mutant, img1, did not grow at all in the presence of 6% ethanol. Kubota et al. (2004) reported 22 genes involved in mitochondrial function that were important for growth on 11% ethanol, indicating that certain mitochondrial functions are important to resist high levels of ethanol. We tested all these mutants at 6% ethanol and found none of them to be sensitive. Remarkably, none of the mitochondrial mutants we identified is reported in the screen of Kubota et al., suggesting that mechanisms of ethanol sensitivity depend on ethanol concentration. We identified 15 mutations in genes involved in vacuolar function and vesicular transport to the vacuole. Three of these mutations are in genes specifically involved in the metabolism of phosphatidyl inositol 3,5-bisphosphate. Genes involved in vacuolar protein sorting and appearing in our screen are represented in all classes of vacuolar morphology except one, class F (Raymond et al., 1992). This strongly suggests the importance of vacuolar function and maintenance under ethanol stress. Kubota et al. lists 45 genes involved in vacuolar function that are important at high ethanol concentrations. We tested all vps mutants and all mutants listed by Kubota et al. Ten of the genes required for growth at 6% ethanol were not picked up in their screen. We identified seven mutants in the PKC1 cell integrity pathway. This pathway is involved in regulation of many responses in yeast cells, including G1–S phase transition, actin polarization/depolarization and the response to cell wall stress (Delley and Hall, 1999). Interestingly, SIT4 and BEM2, which encode proteins involved in the downregulation of signalling through the PKC1 pathway, were essential for growth of yeast on plates containing 6% ethanol. Genes SLG1 and ROM2, encoding proteins involved in upregulation of signalling, are important but not essential for growth in the presence of 6% ethanol (Table 1). We have identified GIM4 and GIM5, encoding subunits of the hetero-oligomeric co-chaperone GimC complex, as important for growth of BY4742 in the presence of 6% ethanol. This may be rationalized as follows. A subset of newly synthesized polypeptides requires folding by a chaperonin. In eukaryotes the formation of native-state tubulin and actin requires folding by the TriC/CCT complex. Efficient folding of proteins by the TriC/CCT complex requires GimC. By interactions with both folding intermediates as well as with TriC/CCT, GimC accelerates actin folding at least five-fold (Siegers et al., 2003). The F-actin dispersion occurring during ethanol stress (Kubota et al., 2004) could be lethal in mutants depleted for components of this complex.

ADA3, GCN5, SPT3 and SPT7 of the SAGA complex are also important for growth of BY4742 in the presence of 6% ethanol. The SAGA (Spt-Ada-Gcn5-histone acetyltransferase) complex is a multi-subunit complex involved in expression of ∼10% of the transcribed part of the genome. It comprises: (a) the Ada proteins (Ada1p, Ada2p, Ada3p, Gcn5p and Ada5p); (b) the Spt proteins (Spt3p, Spt7p, Spt8p and Spt20p); and (c) a number of TAFs (TATA-binding protein associated factors) in addition to the product of the essential TRA1 gene. Both the SAGA complex and TFIID deliver TBP (TATA binding protein) to promoters, leading to gene expression, and both complexes bind TAFs. Whereas TFIID is involved in expression of ∼90% of the expressed genome and is involved in expression of housekeeping genes, the SAGA-controlled genes are largely stress-induced (Huisinga and Pugh, 2004). Our data show the need for components of this complex during ethanol stress, and hence suggests the involvement of a SAGA-dependent response during ethanol stress.

We found 14 mutants that did not belong to any of the mentioned classes. Interestingly, four of these, FEN1, PLC1, ERG6 and SUR4, are involved in lipid biosynthesis, which is in addition to those involved in phosphatidyl inositol 3,5-bisphosphate synthesis (VPS34, VAC14 and FAB1). Lipid metabolism was reported to play a role in response to ethanol stress (Swan and Watson, 1999). fen1 is synthetically lethal with erg6, underlining the importance of the sterol–sphingolipid interaction (Eisenkolb et al., 2002). We tested all other viable mutants in ergosterol biosynthesis but found no strong phenotype at 6% ethanol.

Cross-sensitivity of the mutants

We tested whether the mutants we identified are sensitive to other stresses relevant for brewing conditions. The results are summarized in Table 2, and Figure 2 gives an example of the growth behaviour of three of the mutants. Only three strains showed a growth defect on 0.4 M NaCl, indicating that osmotic problems are in most cases not the reason for the growth defect on 6% ethanol. Nine of the mutants showed a growth defect on sorbic acid plates, suggesting a defect related to acid stress. Ethanol stress has been suggested to resemble temperature stress (Piper, 1995). However, only six of the mutants showed a growth defect at 37 °C, indicating only a limited overlap between ethanol and temperature stress. Several of the mutants we identified are part of the cell wall integrity pathway. Therefore, we tested all our mutants for growth on plates containing the cell wall-perturbing agent Calcofluor white. Interestingly, 15 of the mutants that showed defective growth on 6% ethanol were also sensitive to Calcofluor white. This shows that some of the mechanisms leading to ethanol sensitivity in the deletion strains are related to defects induced in the cell wall. Only rib4, vma10 and vps34 were sensitive to all five types of stress.

thumbnail image

Figure 2. Cross-sensitivity of the wild-type and representative mutants. Ten-fold serial dilutions of the indicated strains were plated on YPD or YPD supplemented with sorbic acid (0.5 mM, pH 4.5) or Calcofluor white (50 µg/ml) and incubated for 5 days at 30 °C or 37 °C, as indicated

Download figure to PowerPoint

Table 2. Sensitivity of ethanol-sensitive deletion strains to other stress conditions
 0.4 M NaCl0.5 mM sorbic acid37 °CCalcofluor white
  1. Growth was scored on YPD in the presence of 0.4 M NaCl, 0.5 mM sorbic acid, pH 4.5, raised temperature (37 °C) or 50 µg/ml Calcofluor white.

No growth RIB4BEM2FAB1
 VPS34ROM2
 SIT4
SLG1
SLT2
SMI1
SWI4
VAC14
VPS34
Reduced growthRIB4BEM2PAT1RIB4
VMA10HMI1RIB4SIN3
VPS34PAT1VMA10VMA10
 RMD8 VPS5
VMA10 VPS30
VPS34 
VPS39 
YDL114W 

Activation of signalling pathways

The transcription factors Msn2p and Msn4p are involved in activation of genes responding to a number of environmental stresses, such as heat shock, osmotic stress and high-ethanol stress (Martinez-Pastor et al., 1996). Deletion of MSN2 alone or MSN4 alone does not result in an ethanol-sensitive phenotype. Deletion of both genes results in a slow-growing, very stress-sensitive strain (data not shown; and Estruch and Carlson, 1993). We made use of a C-terminal GFP-MSN2 fusion protein to determine whether the MSN2/MSN4 pathway is activated under our ethanol stress conditions (Figure 3A). In exponentially growing cells in YPD medium, most fluorescence was found in the cytosol. Half an hour after addition of ethanol to a concentration of 6%, most fluorescence was found in the nucleus, indicating activation of the general stress response. Among the genes that are transcriptionally activated by this general stress pathway, we only found one to be important for growth in the presence of 6% ethanol, viz. the TPS1 gene, encoding trehalose-6-phosphate synthase. A search in the yeast genome resulted in a number of genes with two or more STRE elements in their promoters (Moskvina et al., 1998), but again, among those genes only TPS1 is present in our collection. This suggests that although a wide array of genes is upregulated during ethanol stress by the general stress response pathway, only one of them is important for growth under those conditions.

thumbnail image

Figure 3. Activation of signalling pathways. (A) Localization of Asr1p–GFP in YPD-grown cells and cells in 6% ethanol. (B) Localization of Msn2p–GFP in YPD-grown cells and cells in 6% ethanol. (C) Total amounts of Slt2p (top) and amounts of phosphorylated Slt2p (P-Slt2p) in the indicated mutants. The conditions applied are: 1, YPD; 2, YPD 6% ethanol; 3, YPD 6% ethanol and 1 M sorbitol; 4, YPD with Calcofluor white; 5, YPD with Calcofluor white and 1 M sorbitol

Download figure to PowerPoint

The transcription factor Asr1p responds to the presence of ethanol in the growth medium but not to heat, osmotic and oxidative stresses (Betz et al., 2004). Cells deleted for ASR1 become sensitive to 1% butanol in the growth media but do not show a growth defect in the presence of 1% ethanol. We did not observe a growth defect in the presence of up to 8% ethanol (not shown). The Asr1–GFP fusion protein, however, does respond to the presence of ethanol under the conditions of our screen, by translocating to the nucleus (Figure 3B). The genes under control of Asr1p have not been identified, but either they are not essential for the response to ethanol or their regulation may involve other redundant transcription factors, as is the case for the MSN2/MSN4 pathway.

Several mutations in the cell integrity pathway have strong growth phenotypes in the presence of ethanol. Therefore, we analysed the phosphorylation of the downstream MAP kinase Slt2p. In wild-type (BY4742) cells growing exponentially in YPD, the level of Slt2p phosphorylation is low (Figure 3C, BY4742, 1). Phosphorylation can be induced in the presence of Calcofluor white (Figure 3C, BY4742, 4), which induces cell wall stress. The phosphorylation is absent when the cells are treated with Calcofluor white in the presence of 1 M sorbitol (Figure 3C, BY4742, 5). In the presence of 6% ethanol, phosphorylation of the MAP kinase is also induced, and the stress is likewise relieved by the addition of sorbitol to 1 M (Figure 3C, BY4742, 2, 3). The total expression levels of Stl2p remain the same under all conditions. The high phosphorylation levels in the presence of ethanol could be explained by an inhibitory effect of ethanol on phosphatases that might dephosphorylate Slt2p. However, since sorbitol does not prevent ethanol from entering the cells but does prevent phosphorylation, we do not find this a likely explanation. We therefore propose that the phosphorylation of Slt2p stimulated by ethanol is induced through the cell integrity pathway. This signalling pathway is already known to be involved in many stress responses, e.g. oxidative stress, heat shock, hypo-osmotic stress, actin stress and ER stress (Bonilla and Cunningham, 2003; Harrison et al., 2004; Bryan et al., 2004; Vilella et al., 2005). We decided to analyse Slt2p phosphorylation in three mutants with strong ethanol-sensitive phenotypes. In the bem2 mutant, Slt2p is phosphorylated in response to both Calcofluor white and ethanol, as in the wild-type strain. The total amount of Slt2p is also similar under these conditions (Figure 3C, bem2).

Interestingly, in the smi1 mutant the total amount of Slt2p is significantly higher than in the wild-type (Figure 3C, smi1). The presence of Calcofluor white or ethanol results in a higher phosphorylation than in the wild-type. The higher total amount of Slt2p in the smi1 strain is likely to cause the detected higher amount of phosphorylated Stl2p as compared to the wild-type. Smi1p interacts with Slt2p and is necessary for signalling through the cell integrity pathway (Martin-Yken et al., 2003).

The sit4 mutant behaves similarly to the wild-type but may have slightly lower levels of Slt2p. Slt2p phosphorylation is also low but consistently higher in the presence of ethanol or Calcofluor white (Figure 3C, sit4).

Fermentations

To determine the relevance of our screen for brewery fermentations, we analysed the behaviour of three of the mutants in 200 ml stirred microfermentations. These fermentations can give a first indication for the potential behaviour of yeast strains in larger-scale fermentations. Because BY4742 is not able to utilize maltose, we used complex medium supplemented with 20% glucose. Figure 4 shows the decrease in density of the medium, measured in degrees Plato, over time. The wild-type (BY4742) is able to ferment almost all the sugar in the medium in 70 h. The two mutants in the cell integrity pathway bem2 and sit4 need a substantially longer time to ferment to completeness. Surprisingly, the smi1 mutant ferments just as rapidly as the wild-type. The high expression level of Slt2p may help to overcome the defect in this strain. For two of three strains tested, increased ethanol sensitivity also resulted in impaired high-gravity fermentation. Plate assays for reduced growth on ethanol might give a fair indication for the behaviour of yeast strains under fermentation conditions.

thumbnail image

Figure 4. Stirred high-gravity fermentations of the wild-type and indicated mutants in 200 ml complex medium initially containing 20% glucose

Download figure to PowerPoint

Concluding remarks

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

We identified 46 genes required for efficient growth on 6% ethanol. Under the conditions used, the general stress pathway, the ethanol-specific transcription factor Asr1p and the cell wall integrity pathway are activated. However, mutants lacking the specific target proteins of these pathways were not identified. This suggests that there is a broad response to ethanol involving many components, of which few are absolutely required. Cell wall integrity appears to be a main concern that cells have to deal with under those conditions, given the number of mutants we identified in that pathway and, more importantly, the large number of mutants that showed cross-sensitivity to Calcofluor white. Screens for new mutants with improved fermentation behaviour might be based on the results presented here and focus on the PKC1 pathway and other pathways and genes important for cell wall stability, maintenance and biosynthesis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References

We thank Jette Greisvold for excellent technical assistance and Kjeld Olesen for helpful discussions and advice concerning the small-scale fermentation experiments. This work was funded by the EU Fifth Framework programme ‘Stress-tolerant industrial yeast strains for high-gravity brewing’ (Contract No. QLK1-2001-01066).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Concluding remarks
  7. Acknowledgements
  8. References