Abstract

The first protein map of an ale-fermenting yeast is presented in this paper: 205 spots corresponding to 133 different proteins were identified. Comparison of the proteome of this ale strain with a lager brewing yeast and the Saccharomyces cerevisiae strain S288c confirmed that this ale strain is much closer to S288c than the lager strain at the proteome level. The dynamics of the ale-brewing yeast proteome during production-scale fermentation was analysed at the beginning and end of the first and the third usage of the yeast (called generation in the brewing industry). During the first generation, most changes were related to the switch from aerobic propagation to anaerobic fermentation. Fewer changes were observed during the third generation but certain stress-response proteins such as Hsp26p, Ssa4p and Pnc1p exhibited constitutive expression in subsequent generations. The ale brewing yeast strain appears to be quite well adapted to fermentation conditions and stresses.

Introduction

During fermentation, brewing yeasts are exposed to severe environmental stresses such as variable temperature, high ethanol concentration, high sugar concentration (high-gravity brewing), nutrient limitation, oxygen shortage, and low pH. In general, any environmental factor which could have an adverse effect on cell growth is considered as a stress. A good deal of work on protein and/or mRNA expression has been carried out with laboratory or industrial strains [1–8], but only a few reports have been devoted to lager-brewing yeast transcription under fermentation conditions [9,10] and, to our knowledge, nothing has been published about ale brewing yeast protein expression. Moreover, “top-fermenting” ale and “bottom-fermenting” lager-brewing strains exhibit very different genomic, physiological and fermentation properties. Stress tolerance [40], aroma and flavour formation, fermentation time, reserve carbohydrate accumulation [34] and flocculation capacity [41] vary greatly. Most lager-brewing yeasts are allopolyploid, containing parts of at least two diverged genomes. The first is Saccharamyces cerevisiae, but the second has not yet been clarified despite many investigations focused on this second ancestor. Some homology was reported with S. monacensis[11,12], S. bayanus[13] or S. pastorianus[14,15]. Conversely, ale yeasts are closely related to the S. cerevisiae laboratory strain S288c [16]. Random Amplified PCR (RAPD) analysis has revealed similar patterns between S. cerevisiae strains and the ale yeasts but ale-brewing strains exhibit greater intraspecific heterogeneity than the lager yeasts [16,17]. Amplified fragment length polymorphism (AFLP) analysis of the laboratory and industrial strains also confirmed this pattern of relatedness: 93.7% shared fragments were found between the ale and laboratory strains, but only 74.6% between the lager strain and the same laboratory strains [18]. Two dimensional electrophoresis, a large-scale protein analysis technique, offers great potential for investigating protein expression in a given cell type or cell state and can also provide information on taxonomic relationships between Saccharomyces species [14]. Boucherie and coworkers [19] have pioneered proteomic investigations by 2-D electrophoresis on the budding yeast laboratory strain (S288c). They established a protein reference map with more than 350 identifications. Another protein reference map of a lager brewing strain was recently published by Joubert and coworkers [20] who identified more than 230 proteins.

In this work, we established the first 2-D map of an ale-brewing strain with 205 identified spots corresponding to 133 different proteins. This map will be very helpful for proteomic studies on ale-brewing yeasts. Two-dimensional electrophoresis enabled us to confirm the relatedness between the reference S. cerevisiae S288c and the ale-brewing strains and the discrepancies between these strains and the lager-brewing strain. The aim of this work was:

  • (i) first, to characterise physiological changes occurring during fermentation in the ale yeast, i.e. the adaptation to stressful conditions and the influence of propagation on the first fermentation in order to improve the brewing process,

  • (ii) second, to identify biological markers for the prediction of optimal fermentation progress.

Protein expression was studied at the beginning and the end of the first and the third generation of ale-brewing yeast production-scale fermentation. This study demonstrated great variations related to the switch from respiratory to fermentative metabolism occurring when yeast cells are transferred from propagation to fermentation conditions.

Materials and methods

Strains and culture conditions

Yeast strains used in this study were ale-brewing yeasts A12 and A38 and [14] lager-brewing strains, K3, K6 and K11 as well as S. cerevisiae S288c (Yeast genetics Stock Center, University of California, Berkeley, CA, USA: R.K. Mortimer) used as the reference strain for proteome map comparisons. All comparisons of 2-D maps between ale- and lager-brewing yeasts and the reference S. cerevisiae S288c strain were performed with cells grown in rich YPD medium at 28 °C with agitation (200 rpm). Samples were collected during the exponential growth phase (OD660 nm= 0.6). The A38 ale strain proteome was monitored during fermentation. The industrial fermentation process (Fig. 1) was performed in a 10-hl Tank OutDoor (TOD) pilot device. The A38 ale strain was grown in aerobic conditions with saccharose as the sole carbon source before pitching in wort for the first fermentation (called first generation in brewing terminology). As in the industrial process, the yeasts were harvested at the end of the fermentation and re-inoculated in fresh wort for a second generation. Up to five or six generations can be performed for lager yeasts, many more for ale strains. Proteomic investigations were carried out during the first and third generations. Samples collected at the start, one-quarter and end of both generations were noted F1, F2, F3 (first generation) and T1, T2, T3 (third generation), respectively. Degradation of wort sugars (maltose, glucose, fructose, saccharose and maltotriose) was determined by HPLC (Fig. 1B and C). The A38 ale strain reference map was established on the gel corresponding to the end of the first generation (F3). This gel was chosen as reference since its quality and resolution were the best among all gels performed.

1

(A) Diagram of the industrial fermentation process. (B) Sugar utilisation profile during the first generation. Sampling times for 2-D electrophoresis are indicated by F1, F2 and F3. (C) Sugar utilisation profile during the third generation. Sampling times for 2-D electrophoresis are indicated by T1, T2 and T3. (B) and (D) Strains were cultured as described in Section 2. (◻) – total sugars, (△) – maltose, (▪) – glucose, (▪) – fructose, (x) – saccharose, (+) – maltotriose.

1

(A) Diagram of the industrial fermentation process. (B) Sugar utilisation profile during the first generation. Sampling times for 2-D electrophoresis are indicated by F1, F2 and F3. (C) Sugar utilisation profile during the third generation. Sampling times for 2-D electrophoresis are indicated by T1, T2 and T3. (B) and (D) Strains were cultured as described in Section 2. (◻) – total sugars, (△) – maltose, (▪) – glucose, (▪) – fructose, (x) – saccharose, (+) – maltotriose.

Two-dimensional gel electrophoresis

Protein extractions were carried out as previously described by Boucherie and coworkers [21] from 5 × 107 cells pelleted after centrifugation. After separation, proteins were fixed and visualized using Silver, Coomassie Blue or Sypro Ruby staining. The image acquisition was scanned with UMAX Power LookII for the Silver and Coomassie Blue coloration and with Molecular Imager FX (BioRad) for the Sypro Ruby coloration.

Protein identification

Identifications were performed by MALDI-TOF analysis. Protein spots (randomly selected) were cut out from the gel, and trypsin-digested before mass spectrometry analysis as described by Joubert and coworkers [20]. Monoisotopic peptide masses obtained by MALDI-TOF analysis were used for database searches with the MS-Fit ProteinProspector package (http://prospector.ucsf.edu/ucsfhtml4.0/msfit.htm).

Quantitative gel analysis

Quantitative gel comparisons were performed with BioImage 2-D Analyser Software running on a SUN station. The comparisons were carried out with four Sypro Ruby-stained gels for each stage. In a first step, the software went through a detection and quantification process for each spot of each gel. In a second step, the four Sypro Ruby-stained gels of each stage were matched in a single image gel (called composite) by selecting 20 landmarks, allowing the software to match automatically additional spots. Each individual spot on the 2-D gels was normalized with respect to a normalization factor calculated from total valid spots found on the respective gel. Finally, two composites were also manually landmarked, automatically matched and normalized. The integrated intensity ratios were estimated to search for statistically significant differences in spot intensities between two composites. Ratios greater than 2 (+ or −) were considered significant.

Quantitative sugar analysis

Glucose, fructose, saccharose, maltose and maltotriose in fermentation samples were monitored by HPLC with a La Chrom system (Merck). Samples were injected onto a Shodex NH2P-504E column at 40 °C with acrylonitrile (70%) eluant (flow rate 1 ml min−1).

Resolved sugars were detected by a refractive index detector and quantified with a standard concentration scale.

Results and discussion

Two-dimensional protein map of an industrial ale-brewing yeast

The ale strain A38 was chosen as the reference for establishing a 2-D electrophoresis map of an ale-brewing strain (Fig. 2). The pattern was composed of 1200 polypeptide spots. The first dimension ranged from pH 3.8 to 6.8 and the second dimension from 15 to 180 kDa. Two hundred and fifty spots were selected among the most abundant proteins and excised from Coomassie-blue-stained gel to be identified by MALDI-TOF: 133 different proteins were identified (Table 1). One-third of the identified proteins are involved in amino acid and protein biosynthesis, whereas 23%, 12% and 7.5%, respectively, are involved in carbon metabolism, stress pathways and nucleic acid biosynthesis.

2

Two-dimensional protein reference map of the A38 ale-brewing yeast. The gel presenting the highest quality and resolution was chosen to report protein identifications. The pattern is composed of 1200 polypeptide spots. Spot names followed by an asterisk correspond to protein fragments; 205 spots corresponding to 133 different proteins were identified.

2

Two-dimensional protein reference map of the A38 ale-brewing yeast. The gel presenting the highest quality and resolution was chosen to report protein identifications. The pattern is composed of 1200 polypeptide spots. Spot names followed by an asterisk correspond to protein fragments; 205 spots corresponding to 133 different proteins were identified.

1

List of the 133 proteins identified by MALDI-TOF from the 2-D reference map of an ale-brewing yeast (Fig. 2)

No. pMw (kDa) name ORF name Protein name function 
6.3 58.7 ACH1 YBL015W Acetyl-CoA hydrolase Lipid metabolism 
6.2 75.5 ACS2 YLR153C Acetyl-CoA synthetase 2 To Krebs cycle 
5.4 41.7 ACT1 YFL039C Actin Cytoskeleton 
5.1 86.1 ADE5.7 YGL234W Phosphoribosylamine-glycine ligase and phosphoribosylformylglycinamidine cyclo-ligase Acid nucleic metabolism 
6.1 65.3 ADE16 YLR028C AICAR transformylase/IMP cyclohydrolase Acid nucleic metabolism 
6.1 65.3 ADE17 YMR120C AICAR transformylase/IMP cyclohydrolase Acid nucleic metabolism 
6.7 36.9 ADH1 YOL086c Alcohol dehydrogenase 1 Fermentation 
36.4 ADO1 YJR105W Adenosine kinase Acid nucleic metabolism 
5.4 107 ALA1 YOR335C Alanyl-tRNA synthetase Protein metabolism 
10 6.3 56.7 ALD4 YOR374W Aldehyde dehydrogenase Carbon metabolism 
11 5.3 54.4 ALD6 YPL061W Aldehyde dehydrogenase Carbon metabolism 
12 5.7 38.9 ARA1 YBR149W d-arabinose dehydrogenase Glycolyse/Gluconeogenese 
13 5.5 46.9 ARG1 YOL058W Argininosuccinate synthetase Amino acid metabolism 
14 5.7 56.2 ARO8 YGL202W Aromatic amino acid aminotransferase I Amino acid metabolism 
15 5.7 64.5 ASN1 YPR145W Asparagine synthetase 1 Amino acid metabolism 
16 5.5 54.8 ATP2 YJR121W F1-β ATP synthase Respiration 
17 5.8 34.8 BEL1 YMR116C 40S small subunit ribosomal protein Protein metabolism 
18 4.8 30.1 BMH1 YER177W Homology with 14-3-3 protein Cytoskeleton 
19 4.8 31.1 BMH2 YDR099W Homology with 14-3-3 protein Cytoskeleton 
20 6.5 46.1 CAR2 YLR438W Ornithine aminotransferase Amino acid metabolism 
21 4.9 58.4 CDC48 YDR168W ATPase of AAA family Cellular process 
22 5.9 51.4 CIT2 YCR005C Citrate synthase To Krebs cycle 
23 6.8 50.2 COR1 YBL045C Ubiquinol cytochrome-c reductase core protein 1 Respiration 
24 6.1 42.5 CYS3 YAL012W Cystathionine γ-Lyase Amino acid metabolism 
25 6.3 56 CYS4 YGR155W Cystathionine β-Synthase Amino acid metabolism 
26 6.4 65.3 DLD1 YDL174C d-lactate dehydrogenase To Krebs cycle 
27 6.4 55.2 DLD3 YEL071W Homology with d-lactate dehydrogenase Energy 
28 4.3 22.7 EFB1 YAL003W Translation elongation factor eEF1beta Protein metabolism 
29 5.9 93.3 EFT1 YOR133W Translation elongation factor eEF2 Protein metabolism 
30 6.2 46.8 ENO1 YGR254W Enolase 1 Glycolyse/Gluconeogenese 
31 5.7 46.9 ENO2 YHR174W Enolase 2 Glycolyse/Gluconeogenese 
32 5.5 43.4 ERG6 YML008C S-adenosyl-methionine delta-24-sterol-C-methyltransferase Lipid metabolism 
33 5.5 39.6 FBA1 YKL060C Fructose-bisphosphate aldolase II Glycolyse/Gluconeogenese 
34 5.5 67.4 FRS1 YLR060W Phenylalanyl-tRNA synthetase, alpha subunit Protein metabolism 
35 5.6 57.5 FRS2 YFL022C phenylalanine–tRNA ligase β chain Protein metabolism 
36 8.5 53.1 FUM1 YPL262W Fumarate hydratase Krebs cycle 
37 5.6 49.6 GDH1 YOR375C Glutamate dehydrogenase 1 Amino acid metabolism 
38 5.8 55.4 GLK1 YCL040W Glucokinase Glycolyse/Gluconeogenese 
39 5.9 41.7 GLN1 YPR035W Glutamine synthase Amino acid metabolism 
40 6.2 53.5 GND1 YHR183W 6-Phosphogluconate dehydrogenase Pentose phosphate pathways 
41 5.3 42.9 GPD1 YDL022W Glycerol-3-phosphate dehydrogenase Cellular process 
42 6.1 30.4 GPP1 YIL053W dl-glycerol phosphatase Carbon metabolism 
43 5.8 27.8 GPP2 YER062C dl-glycerol phosphatase Carbon metabolism 
44 5.8 38.2 GRE2 YOL151W Similarity to plant dihydroflavonol-4-reductases Stress 
45 5.7 75.4 GRS1 YBR121C Glycyl-tRNA synthetase Protein metabolism 
46 6.1 24.8 GSP1 YLR293C GTP-binding protein of the ras superfamily Acid nucleic metabolism 
47 6.1 58.5 GUA1 YMR217W GMP synthetase Acid nucleic metabolism 
48 6.3 37.7 HEM13 YDR044W Coproporphyrinogen oxidase Protein metabolism 
49 4.8 80.9 HSC82 YMR186W Heat shock protein Stress 
50 5.3 23.9 HSP26 YBR072W Heat shock protein Stress 
51 4.8 81.4 HSP82 YPL240C Heat shock protein Stress 
52 5.3 102 HSP104 YLL026W Heat shock protein Stress 
53 5.2 60.7 HSP60 YLR259C Heat shock protein Stress 
54 5.2 53.7 HXK1 YFR053C Hexokinase I Glycolyse/Gluconeogenese 
55 5.2 53.9 HXK2 YGL253W Hexokinase II Glycolyse/Gluconeogenese 
56 8.6 74.9 ILV2 YMR108W Acetolactate synthase Amino acid metabolism 
57 11 13.6 ILV5 YLR255C Cetol-acide reductoisomerase Amino acid metabolism 
58 5.4 32.3 IPP1 YBR011C Inorganic pyrophosphatase Phosphate metabolism 
59 4.8 74.5 KAR2 YJL034W Heat shock protein Protein metabolism 
60 6.7 114 KDG1 YIL125W 2-Oxoglutarate dehydrogenase Krebs cycle 
61 8.9 50.4 KGD2 YDR148C 2-Oxoglutarate dehudrogenase Krebs cycle 
62 5.8 67.9 KRS1 YDR037W Lysyl-tRNA synthetase Protein metabolism 
63 7.6 51.8 LAT1 YNL071W Dihydrolipoamide S-acetyltransferase To Krebs cycle 
64 5.5 38.9 LEU2 YCL018W 3-Isopropylmalate dehydrogenase Amino acid metabolism 
65 75.1 LYS4 YDR234W Homoaconitase Amino acid metabolism 
66 6.8 47.1 LYS20 YDL182W Homocitrate synthase Amino acid metabolism 
67 5.1 48.9 LYS9 YNR050C Saccharopine dehydrogenase Amino acid metabolism 
68 5.5 68.1 MAL12 YBR299W Maltase Glycolyse/Gluconeogenese 
69 8.4 74.4 MAE1 YKL029C Malate dehydrogenase Carbon metabolism 
70 5.5 57.7 MET3 YJR010W ATP-sulfurylase Amino acid metabolism 
71 6.1 85.9 MET6 YER091C Homocysteine methyltransferase Amino acid metabolism 
72 48.7 MET25 YLR303W O-acetylhomoserine lyase Amino acid metabolism 
73 5.4 44.9 OYE3 YPL171C NADPH dehydrogenase Energy 
74 5.7 64.3 PAB1 YER165W mRNA polyadenylate-binding protein Acid nucleic metabolism 
75 5.2 40.1 PDB1 YBR221C Pyruvate dehydrogenase To Krebs cycle 
76 5.8 61.5 PDC1 YLR044C Pyruvate decarboxylase 1 Fermentation 
77 9.8 57.7 PDH1 YPR002W Similarity to B. subtilis mmgE protein Unknown 
78 7.8 44.7 PGK1 YCR012W Phosphoglycerate kinase Glycolyse/Gluconeogenese 
79 5.8 24.9 PNC1 YGL037C Nicotinamidase Stress 
80 5.9 69.6 PRB1 YEL060C Protease B, vacuolar Protein metabolism 
81 39.5 PSA1 YDL055C Mannose-1-phosphate guanyltransferase Carbon metabolism 
82 6.4 55.2 PYK2 YOR347C Pyruvate kinase 2 Glycolyse/Gluconeogenese 
83 8.2 23.3 RIP1 YEL024W Ubiquinol-cytochrome C reductase iron–sulfur subunit Respiration 
84 5.1 40.1 RNR4 YGR180C Ribonucleotide reductase small subunit Acid nucleic metabolism 
85 6.4 33.7 RPL1 YPL131W 60S large subunit ribosomal protein Protein metabolism 
86 8.6 25 RPS5 YJR123W Ribosomal protein S5.e Protein metabolism 
87 5.8 49.1 SAH1 YER043C S-adenosyl-l-homocysteine hydrolase Amino acid metabolism 
88 41.8 SAM1 YLR180W S-adenosylmethionine synthetase 1 Amino acid metabolism 
89 5.2 42.3 SAM2 YDR502C S-adenosylmethionine synthetase 2 Amino acid metabolism 
90 5.5 33 SBP1 YHL034C Single-strand nucleic acid binding protein Acid nucleic metabolism 
91 5.9 28 SCL1 YGL011C 20S proteasome subunit YC7ALPHA/Y8 (alpha1) Protein metabolism 
92 5.1 29.1 SEC53 YFL045C Phosphomannomutase Protein metabolism 
93 6.1 43.4 SER1 YOR184W Phosphoserine transaminase Amino acid metabolism 
94 5.8 53.3 SES1 YDR023W Seryl-tRNA synthetase Protein metabolism 
95 5.6 15.9 SOD1 YJR104C Copper–zinc superoxide dismutase Cellular process 
96 8.5 25.8 SOD2 YHR008C Superoxide dismutase Cellular process 
97 5.3 33.3 SPE3 YPR069C Spermidine synthase Amino acid metabolism 
98 69.8 SSA1 YAL005C Heat shock protein of HSP70 family Stress 
99 69.5 SSA2 YLL024C Heat shock protein of HSP70 family Stress 
100 69.7 SSA4 YER103W Heat shock protein of HSP70 family Stress 
101 5.3 66.6 SSB1 YDL229W Heat shock protein of HSP70 family Stress 
102 5.4 66.6 SSB2 YNL209W Heat shock protein of HSP70 family Stress 
103 5.5 70.6 SSC1 YJR045C Mitochondrial heat shock protein 70-related protein Stress 
104 5.1 77.4 SSE1 YPL106C Heat shock protein of HSP70 family Stress 
105 5.5 66.3 STI1 YOR027W Stress-induced protein Stress 
106 6.5 35.8 TDH2 YJR009C Glyceraldehyde-3-phosphate dehydrogenase Glycolyse/Gluconeogenese 
107 6.5 35.8 TDH3 YGR192C Glyceraldehyde-3-phosphate dehydrogenase Glycolyse/Gluconeogenese 
108 6.1 24.3 TFS1 YLR178C CDC25-dependent nutrient- and ammonia-response cell-cycle regulator Cellular process 
109 5.5 57.5 THR4 YCR053W Threonine synthase Amino acid metabolism 
110 44.7 TIF1 YKR059W Translation initiation factor 4A Protein metabolism 
111 5.3 24.3 TIF45 YOL139C Translation initiation factor eIF4E Protein metabolism 
112 5.7 26.8 TPI1 YDR050C Triose-phosphate isomerase Glycolyse/Gluconeogenese 
113 5.7 56.1 TPS1 YBR126C Trehalose-6-phosphate synthase Glycolyse/Gluconeogenese 
114 5.7 34.2 TRR1 YDR353W Thioredoxine-dependante peroxide reductase Cellular process 
115 21.6 TSA1 YML028W Thiol-specifique antioxidant Stress 
116 19.1 TSA2 YLR109W Alkyl hydroperoxide reductase Stress 
117 4.6 50.9 TUB2 YFL037W β-Tubulin Cytoskeleton 
118 6.2 48 TUF1 YOR187W Translation elongation factor TU Protein metabolism 
119 6.3 52.9 UGA1 YGR019W Butyrate transaminase Amino acid metabolism 
120 5.8 34.8 URA1 YKL216W Dihydroorotate dehydrogenase Acid nucleic metabolism 
121 5.8 118 VMA1 YDL185W H+-ATPase V1 domain 69 KD catalytic subunit, vacuolar Cellular process 
122 57.7 VMA2 YBR127C H+-ATPase V1 domain 60 KD subunit, vacuolar Cellular process 
123 5.9 35.6 YDL124W YDL124W Similarity to aldose reductases Unknown 
124 5.3 25.7 YDR533C YDR533C Strong similarity to hypothetical proteins YPL280w. YOR391c and YMR322c Unknown 
125 5.7 116 YEF3 YLR249W Translation elongation factor eEF3 Protein metabolism 
126 5.7 19 YER067W YER067W Strong similarity to hypothetical protein YIL057c Unknown 
127 5.4 52.9 YFR044C YFR044C Similarity to hypothetical protein YBR281c Unknown 
128 5.3 68.6 YGR287C YGR287C Strong similarity to maltase Glycolyse/Gluconeogenese 
129 5.9 44.6 YHB1 YGR234W Flavohemoglobine Cellular process 
130 5.9 27.3 YHR049W YHR049W Similarity to S. pombe dihydrofolate reductase and YOR280c Unknown 
131 4.9 36.7 YIL041W YIL041W Similarity to S. pombe hypothetical protein Unknown 
132 4.7 27.9 YST2 YLR048W 40S ribosomal protein p40 homolog B Protein metabolism 
133 5.7 38.6 YPR127W YPR127W Similarity to C-term, of N. tabacum auxin-induced protein Unknown 
No. pMw (kDa) name ORF name Protein name function 
6.3 58.7 ACH1 YBL015W Acetyl-CoA hydrolase Lipid metabolism 
6.2 75.5 ACS2 YLR153C Acetyl-CoA synthetase 2 To Krebs cycle 
5.4 41.7 ACT1 YFL039C Actin Cytoskeleton 
5.1 86.1 ADE5.7 YGL234W Phosphoribosylamine-glycine ligase and phosphoribosylformylglycinamidine cyclo-ligase Acid nucleic metabolism 
6.1 65.3 ADE16 YLR028C AICAR transformylase/IMP cyclohydrolase Acid nucleic metabolism 
6.1 65.3 ADE17 YMR120C AICAR transformylase/IMP cyclohydrolase Acid nucleic metabolism 
6.7 36.9 ADH1 YOL086c Alcohol dehydrogenase 1 Fermentation 
36.4 ADO1 YJR105W Adenosine kinase Acid nucleic metabolism 
5.4 107 ALA1 YOR335C Alanyl-tRNA synthetase Protein metabolism 
10 6.3 56.7 ALD4 YOR374W Aldehyde dehydrogenase Carbon metabolism 
11 5.3 54.4 ALD6 YPL061W Aldehyde dehydrogenase Carbon metabolism 
12 5.7 38.9 ARA1 YBR149W d-arabinose dehydrogenase Glycolyse/Gluconeogenese 
13 5.5 46.9 ARG1 YOL058W Argininosuccinate synthetase Amino acid metabolism 
14 5.7 56.2 ARO8 YGL202W Aromatic amino acid aminotransferase I Amino acid metabolism 
15 5.7 64.5 ASN1 YPR145W Asparagine synthetase 1 Amino acid metabolism 
16 5.5 54.8 ATP2 YJR121W F1-β ATP synthase Respiration 
17 5.8 34.8 BEL1 YMR116C 40S small subunit ribosomal protein Protein metabolism 
18 4.8 30.1 BMH1 YER177W Homology with 14-3-3 protein Cytoskeleton 
19 4.8 31.1 BMH2 YDR099W Homology with 14-3-3 protein Cytoskeleton 
20 6.5 46.1 CAR2 YLR438W Ornithine aminotransferase Amino acid metabolism 
21 4.9 58.4 CDC48 YDR168W ATPase of AAA family Cellular process 
22 5.9 51.4 CIT2 YCR005C Citrate synthase To Krebs cycle 
23 6.8 50.2 COR1 YBL045C Ubiquinol cytochrome-c reductase core protein 1 Respiration 
24 6.1 42.5 CYS3 YAL012W Cystathionine γ-Lyase Amino acid metabolism 
25 6.3 56 CYS4 YGR155W Cystathionine β-Synthase Amino acid metabolism 
26 6.4 65.3 DLD1 YDL174C d-lactate dehydrogenase To Krebs cycle 
27 6.4 55.2 DLD3 YEL071W Homology with d-lactate dehydrogenase Energy 
28 4.3 22.7 EFB1 YAL003W Translation elongation factor eEF1beta Protein metabolism 
29 5.9 93.3 EFT1 YOR133W Translation elongation factor eEF2 Protein metabolism 
30 6.2 46.8 ENO1 YGR254W Enolase 1 Glycolyse/Gluconeogenese 
31 5.7 46.9 ENO2 YHR174W Enolase 2 Glycolyse/Gluconeogenese 
32 5.5 43.4 ERG6 YML008C S-adenosyl-methionine delta-24-sterol-C-methyltransferase Lipid metabolism 
33 5.5 39.6 FBA1 YKL060C Fructose-bisphosphate aldolase II Glycolyse/Gluconeogenese 
34 5.5 67.4 FRS1 YLR060W Phenylalanyl-tRNA synthetase, alpha subunit Protein metabolism 
35 5.6 57.5 FRS2 YFL022C phenylalanine–tRNA ligase β chain Protein metabolism 
36 8.5 53.1 FUM1 YPL262W Fumarate hydratase Krebs cycle 
37 5.6 49.6 GDH1 YOR375C Glutamate dehydrogenase 1 Amino acid metabolism 
38 5.8 55.4 GLK1 YCL040W Glucokinase Glycolyse/Gluconeogenese 
39 5.9 41.7 GLN1 YPR035W Glutamine synthase Amino acid metabolism 
40 6.2 53.5 GND1 YHR183W 6-Phosphogluconate dehydrogenase Pentose phosphate pathways 
41 5.3 42.9 GPD1 YDL022W Glycerol-3-phosphate dehydrogenase Cellular process 
42 6.1 30.4 GPP1 YIL053W dl-glycerol phosphatase Carbon metabolism 
43 5.8 27.8 GPP2 YER062C dl-glycerol phosphatase Carbon metabolism 
44 5.8 38.2 GRE2 YOL151W Similarity to plant dihydroflavonol-4-reductases Stress 
45 5.7 75.4 GRS1 YBR121C Glycyl-tRNA synthetase Protein metabolism 
46 6.1 24.8 GSP1 YLR293C GTP-binding protein of the ras superfamily Acid nucleic metabolism 
47 6.1 58.5 GUA1 YMR217W GMP synthetase Acid nucleic metabolism 
48 6.3 37.7 HEM13 YDR044W Coproporphyrinogen oxidase Protein metabolism 
49 4.8 80.9 HSC82 YMR186W Heat shock protein Stress 
50 5.3 23.9 HSP26 YBR072W Heat shock protein Stress 
51 4.8 81.4 HSP82 YPL240C Heat shock protein Stress 
52 5.3 102 HSP104 YLL026W Heat shock protein Stress 
53 5.2 60.7 HSP60 YLR259C Heat shock protein Stress 
54 5.2 53.7 HXK1 YFR053C Hexokinase I Glycolyse/Gluconeogenese 
55 5.2 53.9 HXK2 YGL253W Hexokinase II Glycolyse/Gluconeogenese 
56 8.6 74.9 ILV2 YMR108W Acetolactate synthase Amino acid metabolism 
57 11 13.6 ILV5 YLR255C Cetol-acide reductoisomerase Amino acid metabolism 
58 5.4 32.3 IPP1 YBR011C Inorganic pyrophosphatase Phosphate metabolism 
59 4.8 74.5 KAR2 YJL034W Heat shock protein Protein metabolism 
60 6.7 114 KDG1 YIL125W 2-Oxoglutarate dehydrogenase Krebs cycle 
61 8.9 50.4 KGD2 YDR148C 2-Oxoglutarate dehudrogenase Krebs cycle 
62 5.8 67.9 KRS1 YDR037W Lysyl-tRNA synthetase Protein metabolism 
63 7.6 51.8 LAT1 YNL071W Dihydrolipoamide S-acetyltransferase To Krebs cycle 
64 5.5 38.9 LEU2 YCL018W 3-Isopropylmalate dehydrogenase Amino acid metabolism 
65 75.1 LYS4 YDR234W Homoaconitase Amino acid metabolism 
66 6.8 47.1 LYS20 YDL182W Homocitrate synthase Amino acid metabolism 
67 5.1 48.9 LYS9 YNR050C Saccharopine dehydrogenase Amino acid metabolism 
68 5.5 68.1 MAL12 YBR299W Maltase Glycolyse/Gluconeogenese 
69 8.4 74.4 MAE1 YKL029C Malate dehydrogenase Carbon metabolism 
70 5.5 57.7 MET3 YJR010W ATP-sulfurylase Amino acid metabolism 
71 6.1 85.9 MET6 YER091C Homocysteine methyltransferase Amino acid metabolism 
72 48.7 MET25 YLR303W O-acetylhomoserine lyase Amino acid metabolism 
73 5.4 44.9 OYE3 YPL171C NADPH dehydrogenase Energy 
74 5.7 64.3 PAB1 YER165W mRNA polyadenylate-binding protein Acid nucleic metabolism 
75 5.2 40.1 PDB1 YBR221C Pyruvate dehydrogenase To Krebs cycle 
76 5.8 61.5 PDC1 YLR044C Pyruvate decarboxylase 1 Fermentation 
77 9.8 57.7 PDH1 YPR002W Similarity to B. subtilis mmgE protein Unknown 
78 7.8 44.7 PGK1 YCR012W Phosphoglycerate kinase Glycolyse/Gluconeogenese 
79 5.8 24.9 PNC1 YGL037C Nicotinamidase Stress 
80 5.9 69.6 PRB1 YEL060C Protease B, vacuolar Protein metabolism 
81 39.5 PSA1 YDL055C Mannose-1-phosphate guanyltransferase Carbon metabolism 
82 6.4 55.2 PYK2 YOR347C Pyruvate kinase 2 Glycolyse/Gluconeogenese 
83 8.2 23.3 RIP1 YEL024W Ubiquinol-cytochrome C reductase iron–sulfur subunit Respiration 
84 5.1 40.1 RNR4 YGR180C Ribonucleotide reductase small subunit Acid nucleic metabolism 
85 6.4 33.7 RPL1 YPL131W 60S large subunit ribosomal protein Protein metabolism 
86 8.6 25 RPS5 YJR123W Ribosomal protein S5.e Protein metabolism 
87 5.8 49.1 SAH1 YER043C S-adenosyl-l-homocysteine hydrolase Amino acid metabolism 
88 41.8 SAM1 YLR180W S-adenosylmethionine synthetase 1 Amino acid metabolism 
89 5.2 42.3 SAM2 YDR502C S-adenosylmethionine synthetase 2 Amino acid metabolism 
90 5.5 33 SBP1 YHL034C Single-strand nucleic acid binding protein Acid nucleic metabolism 
91 5.9 28 SCL1 YGL011C 20S proteasome subunit YC7ALPHA/Y8 (alpha1) Protein metabolism 
92 5.1 29.1 SEC53 YFL045C Phosphomannomutase Protein metabolism 
93 6.1 43.4 SER1 YOR184W Phosphoserine transaminase Amino acid metabolism 
94 5.8 53.3 SES1 YDR023W Seryl-tRNA synthetase Protein metabolism 
95 5.6 15.9 SOD1 YJR104C Copper–zinc superoxide dismutase Cellular process 
96 8.5 25.8 SOD2 YHR008C Superoxide dismutase Cellular process 
97 5.3 33.3 SPE3 YPR069C Spermidine synthase Amino acid metabolism 
98 69.8 SSA1 YAL005C Heat shock protein of HSP70 family Stress 
99 69.5 SSA2 YLL024C Heat shock protein of HSP70 family Stress 
100 69.7 SSA4 YER103W Heat shock protein of HSP70 family Stress 
101 5.3 66.6 SSB1 YDL229W Heat shock protein of HSP70 family Stress 
102 5.4 66.6 SSB2 YNL209W Heat shock protein of HSP70 family Stress 
103 5.5 70.6 SSC1 YJR045C Mitochondrial heat shock protein 70-related protein Stress 
104 5.1 77.4 SSE1 YPL106C Heat shock protein of HSP70 family Stress 
105 5.5 66.3 STI1 YOR027W Stress-induced protein Stress 
106 6.5 35.8 TDH2 YJR009C Glyceraldehyde-3-phosphate dehydrogenase Glycolyse/Gluconeogenese 
107 6.5 35.8 TDH3 YGR192C Glyceraldehyde-3-phosphate dehydrogenase Glycolyse/Gluconeogenese 
108 6.1 24.3 TFS1 YLR178C CDC25-dependent nutrient- and ammonia-response cell-cycle regulator Cellular process 
109 5.5 57.5 THR4 YCR053W Threonine synthase Amino acid metabolism 
110 44.7 TIF1 YKR059W Translation initiation factor 4A Protein metabolism 
111 5.3 24.3 TIF45 YOL139C Translation initiation factor eIF4E Protein metabolism 
112 5.7 26.8 TPI1 YDR050C Triose-phosphate isomerase Glycolyse/Gluconeogenese 
113 5.7 56.1 TPS1 YBR126C Trehalose-6-phosphate synthase Glycolyse/Gluconeogenese 
114 5.7 34.2 TRR1 YDR353W Thioredoxine-dependante peroxide reductase Cellular process 
115 21.6 TSA1 YML028W Thiol-specifique antioxidant Stress 
116 19.1 TSA2 YLR109W Alkyl hydroperoxide reductase Stress 
117 4.6 50.9 TUB2 YFL037W β-Tubulin Cytoskeleton 
118 6.2 48 TUF1 YOR187W Translation elongation factor TU Protein metabolism 
119 6.3 52.9 UGA1 YGR019W Butyrate transaminase Amino acid metabolism 
120 5.8 34.8 URA1 YKL216W Dihydroorotate dehydrogenase Acid nucleic metabolism 
121 5.8 118 VMA1 YDL185W H+-ATPase V1 domain 69 KD catalytic subunit, vacuolar Cellular process 
122 57.7 VMA2 YBR127C H+-ATPase V1 domain 60 KD subunit, vacuolar Cellular process 
123 5.9 35.6 YDL124W YDL124W Similarity to aldose reductases Unknown 
124 5.3 25.7 YDR533C YDR533C Strong similarity to hypothetical proteins YPL280w. YOR391c and YMR322c Unknown 
125 5.7 116 YEF3 YLR249W Translation elongation factor eEF3 Protein metabolism 
126 5.7 19 YER067W YER067W Strong similarity to hypothetical protein YIL057c Unknown 
127 5.4 52.9 YFR044C YFR044C Similarity to hypothetical protein YBR281c Unknown 
128 5.3 68.6 YGR287C YGR287C Strong similarity to maltase Glycolyse/Gluconeogenese 
129 5.9 44.6 YHB1 YGR234W Flavohemoglobine Cellular process 
130 5.9 27.3 YHR049W YHR049W Similarity to S. pombe dihydrofolate reductase and YOR280c Unknown 
131 4.9 36.7 YIL041W YIL041W Similarity to S. pombe hypothetical protein Unknown 
132 4.7 27.9 YST2 YLR048W 40S ribosomal protein p40 homolog B Protein metabolism 
133 5.7 38.6 YPR127W YPR127W Similarity to C-term, of N. tabacum auxin-induced protein Unknown 

The Mw and pI of each protein are those given in the MIPS catalogue.

Two or more spots were identified for several proteins. For some, the only difference was a change in pI, generally due to post-translational modifications. The others displayed changes in both pI and molecular weight, which would probably correspond to protein fragments generated by vacuolar or proteasome degradation [22]. Moreover, about 20% of proteins present on the 2-D gels have been shown to be N-acetylated by an N-terminal acetyltransferase which leads to a shift in their pI position [19,23]. Other post-translational modifications, such as phosphorylation, glycosylation, lipidation and sulfation [24–27], could be identified on the 2-D gel. As also observed by Larsen and coworkers [28], Eno2p was represented by several spots, probably corresponding to different C- and/or N-terminal processed forms. Finally, 21 identified spots appeared to be protein fragments since their molecular weight was smaller than that of the corresponding protein, whereas the covering percentage ranged between 14% and 50%. Most of them belong to the glycolytic pathway. But in general the shift in spot position, for most of the proteins, remains unexplained.

Qualitative comparison between proteomes of the ale- and lager-brewing yeasts, and the reference strain S. cerevisiae S288c

It has previously been reported that 2-D electrophoresis can be used to define the relatedness between yeast strains [14,29]. Therefore, 2-D protein patterns of three yeast strains, the ale A38 ‘top-fermenting’ brewing strain, the K11 ‘bottom-fermenting’ lager-brewing strain, and S. cerevisiae S288c were compared (Fig. 3). All were grown on rich medium, in respiro-fermentative conditions and harvested during the exponential phase. Whereas protein patterns of A38 and S288c appeared similar, the pattern of K11 differed from those of A38 and S288c. This is consistent with the fact that A38 has been described as a tetraploide of S. cerevisiae whereas K11 is a hybrid made up of at least two different genomes: one derived from S. cerevisiae and the other still not clearly identified. Fig. 4 presents detail of patterns of three different lager strains (A–C), S. cerevisiae S288c (D), and two different ale strains (E and F). Previous co-migration experiments between S. cerevisiae S288c and the K11 strains performed by Joubert and coworkers [14] have shown that spots a′, b′ and c′, identified by micro-sequencing as Pdc1p, Eno2p and Fba1, respectively, correspond to S. cerevisiae proteins. Spots a, b and c were, however, identified on K11 strains as isoforms of respectively a′, b′ and c′ and as belonging to S. pastorianus NRRL Y-1551. The pattern of the “second parental strain” appeared clearly on the 2-D gel of K11, but not on the A38 ale strain gel (Fig. 4F). Consequently, the A38 ale strain exhibited the same pattern as the S. cerevisiae strain. These observations can be extended to other ale and lager strains. For example, another ale strain called A12 (Fig. 4E) displayed a protein pattern similar to that of the A38 ale strain (Fig. 4F). Protein patterns of the lager yeasts K3 (Fig. 4B), K6 (Fig. 4C) and K11 (Fig. 4A) also displayed common patterns.

3

Comparison of the 2-D gel electrophoresis pattern between an ale brewing strain, a lager-brewing strain and a laboratory strain. Sypro Ruby protein staining. (A) A38 ale-brewing yeast, (B) S. cerevisiae S288c reference strain and (C) K11 lager-brewing yeast. a′, b′, c′ and their isoforms a, b, c were identified as Pdc1p, Eno2p and Fba1p, respectively.

3

Comparison of the 2-D gel electrophoresis pattern between an ale brewing strain, a lager-brewing strain and a laboratory strain. Sypro Ruby protein staining. (A) A38 ale-brewing yeast, (B) S. cerevisiae S288c reference strain and (C) K11 lager-brewing yeast. a′, b′, c′ and their isoforms a, b, c were identified as Pdc1p, Eno2p and Fba1p, respectively.

4

Two-dimensional gel electrophoresis patterns of ale- and lager-brewing yeasts and of a laboratory strain. Proteins were revealed by silver staining. (A) K11 lager- brewing yeast, (B) K3 lager-brewing yeast, (C) K6 lager-brewing yeast, (D) S. cerevisiae S288c reference strain, (E) A12 ale-brewing yeast and (F) A38 ale-brewing yeast. a′, b′, c′ and their isoforms a, b, c were identified as Pdc1p, Eno2p and Fba1p, respectively.

4

Two-dimensional gel electrophoresis patterns of ale- and lager-brewing yeasts and of a laboratory strain. Proteins were revealed by silver staining. (A) K11 lager- brewing yeast, (B) K3 lager-brewing yeast, (C) K6 lager-brewing yeast, (D) S. cerevisiae S288c reference strain, (E) A12 ale-brewing yeast and (F) A38 ale-brewing yeast. a′, b′, c′ and their isoforms a, b, c were identified as Pdc1p, Eno2p and Fba1p, respectively.

Protein identifications performed for S288c and A38 reference maps confirmed that their genomes are closely related. However, this does not mean that these strains are identical, as the comparison was performed only on a detail part of the map. When looking at the entire gel for both strains, some discrepancies appear. For example, Adh4p is present in the brewing strain but not in S288c (Fig. 3). Adh4 expression seems to be specific for the brewing strain in such conditions. To summarize, 2-D electrophoresis is a powerful technology for confirming genetic background and distinguishing between ale- and lager-brewing strains, and for monitoring protein expression during a fermentation process.

Protein expression in ale strain A38 during fermentation

After aerobic propagation, yeasts were inoculated in wort for the first generation. During that generation, cells replicated two to four times. Yeasts were then harvested and re-used for inoculating the next generation (Fig. 1). Two-dimensional gels were obtained with protein extracts collected at the beginning, one quarter and the end of the first generation and at the end of the third re-used cell generation. Fig. 1B and C indicate the sampling times on sugar consumption curves (F1, F2, F3 and T1, T2, T3). Propagation for the first generation was carried out under aerobic conditions and on synthetic medium containing saccharose as the sole carbon source. Qualitative 2-D gel comparisons showed few variations between the samples harvested at the beginning (F1, T1) and at the first quarter (F2, T2) of the generations. Whereas samples collected at the end of generations (T2/T3), compared to the first quarter samples (F2/F3), present similar protein variations as those observed in the comparison between the beginning and the end of the generations (F1/F3, T1/T3). As no supplementary information was presented on the F2 and T2 step gels, quantitative analysis was only performed on gels corresponding to the beginning (F1,T1) and the end (F3,T3) stages of the first and third fermentations. Ratios greater than 2 (+ or −) were considered significant. All spots that exhibited significant intensity differences were subjected to MALDI-TOF identification. Some spots with weak intensity gave few peptide mass peaks, which were insufficient for unambiguous identification of the protein. However, the identification rate reached 70% and revealed that the first generation displayed the greatest number of changes, with 85 significant changes in comparison with 27 during the third generation. Results are reported in Table 2 and Fig. 5A for the first generation, and Table 3 and Fig. 5B for the third generation.

2

List of decreased or increased quantities of proteins from the comparison of the beginning (F1) and the end (F3) of the first generation

No. pMw ORF name Gene or ORF name % Coveragea Factor Name or function Group functions 
69.7 YER103w SSA4 29 Present Heat shock protein of HSP70 family, cytosolic Stress 
4.8 74.5 YJL034w KAR2 44 Present Nuclear fusion protein Stress 
– – NI NI NI Present NI NI 
6.3 37.7 YDR044w HEM13 29 Present Coproporphyrinogen III oxidase Protein biosynthesis 
– – NI NI NI Present NI NI 
– – NI NI NI Present NI NI 
– – NI NI NI Present NI NI 
– – NI NI NI Present NI NI 
5.8 25 YGL037c PNC1 37 Present Similarity to PIR:B70386 pyrazinamidase/nicotinamidase - Aquifex aeolicus Unknown 
10 – – NI NI NI Present NI NI 
11 5.3 23.9 YBR072w HSP26∗ 39 4.4 Heat shock protein Stress 
12 – – NI NI NI 4.113 NI NI 
13 5.7 38.6 YPR127w YPR127w 36 3.79 Similarity to C-term, of N.tabacum auxin-induced protein Unknown 
14 6.3 37.7 YDR044w HEM13 29 3.75 Coproporphyrinogen III oxidase Protein biosynthesis 
15 5.3 25.7 YDR533c YDR533c 29 3.47 Hypothetical proteins YPL280w, YOR391c and YMR322c Unknown 
16 5.8 61.5 YLR044c PDC1∗ 23 3.44 Pyruvate decarboxylase, isozyme 1 Carbon metabolism 
17 6.1 24.4 YLR178c TFS1 30 3.37 Nutrient- and ammonia-response cell-cycle regulator Unknown 
18 – – NI NI NI 3.104 NI NI 
19 – – NI NI NI 3.09 NI NI 
20 – – NI NI NI 2.94 NI NI 
21 5.3 23.9 YBR072w HSP26 39 2.78 Heat shock protein Stress 
22 – – YGR254w/YHR174w ENO∗ 18/21 2.73 Enolase 1 or enolase 2 Carbon metabolism 
23 – – NI NI NI 2.65 NI NI 
24 7.1 44.7 YCR012w PGK1∗ 28 2.6 Phosphoglycerate kinase Carbon metabolism 
25 – – NI NI NI 2.5 NI NI 
26 – – NI NI NI 2.48 NI NI 
27 5.5 39.6 YKL060c FBA1∗ 17 2.41 Fructose-bisphosphate aldolase Carbon metabolism 
28 6.4 55.2 YOR347c PYK2∗ 14 2.24 Pyruvate kinase, glucose-repressed isoform Carbon metabolism 
29 – – NI NI NI 2.19 NI NI 
30 6.5 46.1 YLR438w CAR2 35 2.17 Ornithine aminotransferase Amino acid biosynthesis 
31 – – YGR254w /YHR174w ENO 50/50 2.168 Enolase 1 or enolase 2 Carbon metabolism 
32 – – NI NI NI 2.12 NI NI 
33 – – NI NI NI 2.11 NI NI 
34 5.8 61.5 YLR044c PDC1∗ 2.09 Pyruvate decarboxylase, isozyme 1 Carbon metabolism 
35 – – NI NI NI 2.04 NI NI 
36 5.5 70.6 YJR045c SSC1 30 −2.01 Mitochondrial heat shock protein 70-related protein Stress 
37 – – NI NI NI −2.07 NI NI 
38 4.9 36.7 YIL041w YIL041w 26 −2.09 Similarity to S. pombe hypothetical protein Unknown 
39 6.7 114 YIL125w KGD1 22 −2.1 2-oxoglutarate dehydrogenase complex E1 component Unknown 
40 5.2 42.3 YDR502c SAM2 31 −2.1 S-adenosylmethionine synthetase 2 Amino acid biosynthesis 
41 8.5 53.2 YPL262w FUM1 10 −2.11 Fumarate hydratase Carbon metabolism 
42 – – NI NI NI −2.11 NI NI 
43 5.7 38.9 YBR149w ARA1 50 −2.17 d-arabinose dehydrogenase, large subunit Carbon metabolism 
44 6.2 48 YOR187w TUF1 43 −2.2 Translation elongation factor TU, mitochondrial Protein biosynthesis 
45 6.1 58.5 YMR217w GUA1 22 −2.22 GMP synthase (glutamine-hydrolyzing) Nucleotides biosynthesis 
46 5.5 43.4 YML008c ERG6 16 −2.22 S-adenosyl-methionine delta-24-sterol-c-methyltransferase Lipid biosynthesis 
47 – – NI NI NI −2.23 NI NI 
48 5.8 55.4 YCL040w GLK1 28 −2.26 Aldohexose specific glucokinase Carbon metabolism 
49 5.3 33.3 YPR069c SPE3 −2.3 Putrescine aminopropyltransferase (spermidine synthase) Amino acid biosynthesis 
50 5.5 54.8 YJR121w ATP2 36 −2.32 F1F0-ATPase complex, F1 β subunit Respiration 
51 – – NI NI NI −2.38 NI NI 
52 6.5 35.7 YGR192c TDH3 56 −2.4 Glyceraldehyde-3-phosphate dehydrogenase 3 Carbon metabolism 
53 6.2 36.8 YOL086c ADH1 40 −2.41 Alcohol dehydrogenase I Carbon metabolism 
54 48.7 YLR303w MET17 41 −2.42 O-acetylhomoserine sulfhydrylase Amino acid biosynthesis 
55 36.4 YJR105w ADO1 39 −2.48 Strong similarity to human adenosine kinase Nucleotides biosynthesis 
56 5.9 44.6 YGR234w YHB1 18 −2.51 Flavohemoglobin Detoxification 
57 – – NI NI NI −2.51 NI NI 
58 5.2 60.8 YLR259c HSP60 34 −2.53 Heat shock protein – chaperone, mitochondrial Stress 
59 6.2 36.8 YOL086c ADH1 40 −2.56 Alcohol dehydrogenase I Carbon metabolism 
60 6.2 36.8 YOL086c ADH1 40 −2.6 Alcohol dehydrogenase I Carbon metabolism 
61 5.5 70.6 YJR045c SSC1 30 −2.62 Mitochondrial heat shock protein 70-related protein Stress 
62 6.3 56.7 YOR374w ALD4 36 −2.62 Aldehyde dehydrogenase, mitochondrial Carbon metabolism 
63 9.1 44.4 YLR355c ILV5 47 −2.63 Ketol-acid reducto-isomerase Amino acid biosynthesis 
64 5.5 46.9 YOL058w ARG1 14 −2.74 Argininosuccinate synthetase Amino acid biosynthesis 
65 5.5 43.4 YML008c ERG6 16 −2.87 S-adenosyl-methionine delta-24-sterol-c-methyltransferase Lipid biosynthesis 
66 5.5 70.6 YJR045c SSC1 30 −2.88 Mitochondrial heat shock protein 70-related protein Stress 
67 8.9 50.4 YDR148c KGD2 22 −3.11 2-oxoglutarate dehydrogenase complex E2 component Carbon metabolism 
68 5.3 68.6 YGR287c YGR287c 32 −3.22 Strong similarity to maltase Unknown 
69 5.3 53.7 YFR053c HXK1 12 −3.36 Hexokinase I Carbon metabolism 
70 6.1 85.9 YER091c MET6 38 −3.49 Homocysteine methyltransferase Amino acid biosynthesis 
71 6.1 85.9 YER091c MET6∗ 38 −3.5 Homocysteine methyltransferase Amino acid biosynthesis 
72 5.2 53.9 YGL253w HXK2 33 −3.57 Hexokinase II Carbon metabolism 
73 8.2 23.4 YEL024w RIP1 26 −3.59 Ubiquinol–cytochrome-c reductase iron-sulfur protein precursor Respiration 
74 5.3 53.7 YFR053c HXK1 12 −3.64 Hexokinase I Carbon metabolism 
75 5.5 54.8 YJR121w ATP2 36 −3.67 F1F0-ATPase complex, F1 β subunit Respiration 
76 5.2 42.3 YDR502c SAM2 31 −4.09 S-adenosylmethionine synthetase 2 Amino acid biosynthesis 
77 5.5 54.8 YJR121w ATP2 36 −4.1 F1F0-ATPase complex, F1 β subunit Respiration 
78 41.8 YLR180w SAM1 30 −4.15 S-adenosylmethionine synthetase 1 Amino acid biosynthesis 
79 5.3 54.4 YPL061w ALD6 17 −4.73 Aldehyde dehydrogenase, cytosolic Carbon metabolism 
80 6.8 50.2 YBL045c COR1 39 −5.09 Ubiquinol—cytochrome-c reductase 44 K core protein Respiration 
81 5.9 51.4 YCR005c CIT2 18 −5.37 Citrate (si)-synthase, peroxisomal/Ubiquinol–cytochrome-c reductase 44K core protein Respiration 
82 5.9 44.6 YGR234w YHB1 18 −5.71 flavohemoprotein Detoxification 
83 6.3 58.7 YBL015w ACH1 46 −6.26 Acetyl-CoA hydrolase Carbon metabolism 
84 5.8 68 YDR037w KRS1 16 −10.56 Lysyl-tRNA synthetase, cytosolic Protein biosynthesis 
85 5.2 42.3 YDR502c SAM2 31 Absent S-adenosylmethionine synthetase 2 Amino acid biosynthesis 
No. pMw ORF name Gene or ORF name % Coveragea Factor Name or function Group functions 
69.7 YER103w SSA4 29 Present Heat shock protein of HSP70 family, cytosolic Stress 
4.8 74.5 YJL034w KAR2 44 Present Nuclear fusion protein Stress 
– – NI NI NI Present NI NI 
6.3 37.7 YDR044w HEM13 29 Present Coproporphyrinogen III oxidase Protein biosynthesis 
– – NI NI NI Present NI NI 
– – NI NI NI Present NI NI 
– – NI NI NI Present NI NI 
– – NI NI NI Present NI NI 
5.8 25 YGL037c PNC1 37 Present Similarity to PIR:B70386 pyrazinamidase/nicotinamidase - Aquifex aeolicus Unknown 
10 – – NI NI NI Present NI NI 
11 5.3 23.9 YBR072w HSP26∗ 39 4.4 Heat shock protein Stress 
12 – – NI NI NI 4.113 NI NI 
13 5.7 38.6 YPR127w YPR127w 36 3.79 Similarity to C-term, of N.tabacum auxin-induced protein Unknown 
14 6.3 37.7 YDR044w HEM13 29 3.75 Coproporphyrinogen III oxidase Protein biosynthesis 
15 5.3 25.7 YDR533c YDR533c 29 3.47 Hypothetical proteins YPL280w, YOR391c and YMR322c Unknown 
16 5.8 61.5 YLR044c PDC1∗ 23 3.44 Pyruvate decarboxylase, isozyme 1 Carbon metabolism 
17 6.1 24.4 YLR178c TFS1 30 3.37 Nutrient- and ammonia-response cell-cycle regulator Unknown 
18 – – NI NI NI 3.104 NI NI 
19 – – NI NI NI 3.09 NI NI 
20 – – NI NI NI 2.94 NI NI 
21 5.3 23.9 YBR072w HSP26 39 2.78 Heat shock protein Stress 
22 – – YGR254w/YHR174w ENO∗ 18/21 2.73 Enolase 1 or enolase 2 Carbon metabolism 
23 – – NI NI NI 2.65 NI NI 
24 7.1 44.7 YCR012w PGK1∗ 28 2.6 Phosphoglycerate kinase Carbon metabolism 
25 – – NI NI NI 2.5 NI NI 
26 – – NI NI NI 2.48 NI NI 
27 5.5 39.6 YKL060c FBA1∗ 17 2.41 Fructose-bisphosphate aldolase Carbon metabolism 
28 6.4 55.2 YOR347c PYK2∗ 14 2.24 Pyruvate kinase, glucose-repressed isoform Carbon metabolism 
29 – – NI NI NI 2.19 NI NI 
30 6.5 46.1 YLR438w CAR2 35 2.17 Ornithine aminotransferase Amino acid biosynthesis 
31 – – YGR254w /YHR174w ENO 50/50 2.168 Enolase 1 or enolase 2 Carbon metabolism 
32 – – NI NI NI 2.12 NI NI 
33 – – NI NI NI 2.11 NI NI 
34 5.8 61.5 YLR044c PDC1∗ 2.09 Pyruvate decarboxylase, isozyme 1 Carbon metabolism 
35 – – NI NI NI 2.04 NI NI 
36 5.5 70.6 YJR045c SSC1 30 −2.01 Mitochondrial heat shock protein 70-related protein Stress 
37 – – NI NI NI −2.07 NI NI 
38 4.9 36.7 YIL041w YIL041w 26 −2.09 Similarity to S. pombe hypothetical protein Unknown 
39 6.7 114 YIL125w KGD1 22 −2.1 2-oxoglutarate dehydrogenase complex E1 component Unknown 
40 5.2 42.3 YDR502c SAM2 31 −2.1 S-adenosylmethionine synthetase 2 Amino acid biosynthesis 
41 8.5 53.2 YPL262w FUM1 10 −2.11 Fumarate hydratase Carbon metabolism 
42 – – NI NI NI −2.11 NI NI 
43 5.7 38.9 YBR149w ARA1 50 −2.17 d-arabinose dehydrogenase, large subunit Carbon metabolism 
44 6.2 48 YOR187w TUF1 43 −2.2 Translation elongation factor TU, mitochondrial Protein biosynthesis 
45 6.1 58.5 YMR217w GUA1 22 −2.22 GMP synthase (glutamine-hydrolyzing) Nucleotides biosynthesis 
46 5.5 43.4 YML008c ERG6 16 −2.22 S-adenosyl-methionine delta-24-sterol-c-methyltransferase Lipid biosynthesis 
47 – – NI NI NI −2.23 NI NI 
48 5.8 55.4 YCL040w GLK1 28 −2.26 Aldohexose specific glucokinase Carbon metabolism 
49 5.3 33.3 YPR069c SPE3 −2.3 Putrescine aminopropyltransferase (spermidine synthase) Amino acid biosynthesis 
50 5.5 54.8 YJR121w ATP2 36 −2.32 F1F0-ATPase complex, F1 β subunit Respiration 
51 – – NI NI NI −2.38 NI NI 
52 6.5 35.7 YGR192c TDH3 56 −2.4 Glyceraldehyde-3-phosphate dehydrogenase 3 Carbon metabolism 
53 6.2 36.8 YOL086c ADH1 40 −2.41 Alcohol dehydrogenase I Carbon metabolism 
54 48.7 YLR303w MET17 41 −2.42 O-acetylhomoserine sulfhydrylase Amino acid biosynthesis 
55 36.4 YJR105w ADO1 39 −2.48 Strong similarity to human adenosine kinase Nucleotides biosynthesis 
56 5.9 44.6 YGR234w YHB1 18 −2.51 Flavohemoglobin Detoxification 
57 – – NI NI NI −2.51 NI NI 
58 5.2 60.8 YLR259c HSP60 34 −2.53 Heat shock protein – chaperone, mitochondrial Stress 
59 6.2 36.8 YOL086c ADH1 40 −2.56 Alcohol dehydrogenase I Carbon metabolism 
60 6.2 36.8 YOL086c ADH1 40 −2.6 Alcohol dehydrogenase I Carbon metabolism 
61 5.5 70.6 YJR045c SSC1 30 −2.62 Mitochondrial heat shock protein 70-related protein Stress 
62 6.3 56.7 YOR374w ALD4 36 −2.62 Aldehyde dehydrogenase, mitochondrial Carbon metabolism 
63 9.1 44.4 YLR355c ILV5 47 −2.63 Ketol-acid reducto-isomerase Amino acid biosynthesis 
64 5.5 46.9 YOL058w ARG1 14 −2.74 Argininosuccinate synthetase Amino acid biosynthesis 
65 5.5 43.4 YML008c ERG6 16 −2.87 S-adenosyl-methionine delta-24-sterol-c-methyltransferase Lipid biosynthesis 
66 5.5 70.6 YJR045c SSC1 30 −2.88 Mitochondrial heat shock protein 70-related protein Stress 
67 8.9 50.4 YDR148c KGD2 22 −3.11 2-oxoglutarate dehydrogenase complex E2 component Carbon metabolism 
68 5.3 68.6 YGR287c YGR287c 32 −3.22 Strong similarity to maltase Unknown 
69 5.3 53.7 YFR053c HXK1 12 −3.36 Hexokinase I Carbon metabolism 
70 6.1 85.9 YER091c MET6 38 −3.49 Homocysteine methyltransferase Amino acid biosynthesis 
71 6.1 85.9 YER091c MET6∗ 38 −3.5 Homocysteine methyltransferase Amino acid biosynthesis 
72 5.2 53.9 YGL253w HXK2 33 −3.57 Hexokinase II Carbon metabolism 
73 8.2 23.4 YEL024w RIP1 26 −3.59 Ubiquinol–cytochrome-c reductase iron-sulfur protein precursor Respiration 
74 5.3 53.7 YFR053c HXK1 12 −3.64 Hexokinase I Carbon metabolism 
75 5.5 54.8 YJR121w ATP2 36 −3.67 F1F0-ATPase complex, F1 β subunit Respiration 
76 5.2 42.3 YDR502c SAM2 31 −4.09 S-adenosylmethionine synthetase 2 Amino acid biosynthesis 
77 5.5 54.8 YJR121w ATP2 36 −4.1 F1F0-ATPase complex, F1 β subunit Respiration 
78 41.8 YLR180w SAM1 30 −4.15 S-adenosylmethionine synthetase 1 Amino acid biosynthesis 
79 5.3 54.4 YPL061w ALD6 17 −4.73 Aldehyde dehydrogenase, cytosolic Carbon metabolism 
80 6.8 50.2 YBL045c COR1 39 −5.09 Ubiquinol—cytochrome-c reductase 44 K core protein Respiration 
81 5.9 51.4 YCR005c CIT2 18 −5.37 Citrate (si)-synthase, peroxisomal/Ubiquinol–cytochrome-c reductase 44K core protein Respiration 
82 5.9 44.6 YGR234w YHB1 18 −5.71 flavohemoprotein Detoxification 
83 6.3 58.7 YBL015w ACH1 46 −6.26 Acetyl-CoA hydrolase Carbon metabolism 
84 5.8 68 YDR037w KRS1 16 −10.56 Lysyl-tRNA synthetase, cytosolic Protein biosynthesis 
85 5.2 42.3 YDR502c SAM2 31 Absent S-adenosylmethionine synthetase 2 Amino acid biosynthesis 

“Present” as factor indication indicates that the spot was observed only on the end of fermentation. Inversely “absent” as factor indication indicates that the spot was missing at the end of fermentation.

a

Percentage of matched peptide covering the protein obtained after spot identification by mass spectrometry and MS-Fit questioning. Spot identifications with percentage coverage below 10% were confirmed by Q-TOF analysis. The Mw and pI of each protein are those given in the MIPS catalogue. NI indicates that a spot was not identified by MALDI-TOF. These proteins are reported by their numbers in Fig. 5A.

5

Comparison between the beginning and the end of the first and the third generations. Proteins were revealed by Sypro Ruby staining and quantitatively compared by BioImage software. Only proteins of variable expression are annotated. Spot intensities increasing more than 2-fold are underlined whereas intensities decreasing less than 2-fold are not. Protein names followed by an asterisk designate fragments. Spots indicated by a number were also variable but not identified by MALDI-TOF. (A) First fermentation: 2-D gel electrophoresis comparison between (a) the beginning (F1) and (b) the end (F3). (B) Third fermentation: 2-D gel electrophoresis comparison between (a) the beginning (T1) and (b) the end (T3).

5

Comparison between the beginning and the end of the first and the third generations. Proteins were revealed by Sypro Ruby staining and quantitatively compared by BioImage software. Only proteins of variable expression are annotated. Spot intensities increasing more than 2-fold are underlined whereas intensities decreasing less than 2-fold are not. Protein names followed by an asterisk designate fragments. Spots indicated by a number were also variable but not identified by MALDI-TOF. (A) First fermentation: 2-D gel electrophoresis comparison between (a) the beginning (F1) and (b) the end (F3). (B) Third fermentation: 2-D gel electrophoresis comparison between (a) the beginning (T1) and (b) the end (T3).

3

List of decreased or increased quantities of proteins from the comparison of the beginning (T1) and the end (T3) of the third generation

No. pMw (kDa) ORF name Gene or ORF name % Coveragea Factor Name or function Group functions 
5.8 49.1 YER043c SAH1 24 5.5 S-adenosyl-l-homocysteine hydrolase Amino acid biosynthesis 
69.8 YAL005c SSA1 43 4.96 Heat shock protein of HSP70 family, cytosolic Stress 
9.2 57.7 YPR002w PDH1∗ 43 3.77 Similarity to B. subtilis mmgE protein Carbon metabolism 
5.5 54.8 YJR121w ATP2∗ 23 3.53 F1F0-ATPase complex, F1 β subunit Respiration 
4.8 74.5 YJL034w KAR2 44 3.35 Nuclear fusion protein Stress 
5.5 39.6 YKL060c FBA1∗ 17 2.78 Fructose-bisphosphate aldolase Carbon metabolism 
5.8 61.5 YLR044c PDC1 26 2.65 Pyruvate decarboxylase, isozyme 1 Carbon metabolism 
– – NI NI NI 2.61 NI NI 
– – NI NI NI 2.55 NI NI 
10 6.2 36.8 YOL086c ADH1 26 2.53 Alcohol dehydrogenase I Carbon metabolism 
11 69.8 YAL005c SSA1 28 2.48 Heat shock protein of HSP70 family, cytosolic Stress 
12 6.2 53.5 YHR183w GND1 34 2.45 6-phosphogluconate dehydrogenase Carbon metabolism 
13 4.6 50.9 YFL037w TUB2 29 2.41 β-Tubulin Cytoskeleton 
14 – – NI NI NI 2.38 NI NI 
15 – – NI NI NI 2.35 NI NI 
16 5.4 44.9 YPL171c OYE3 11 2.35 NAPDH dehydrogenase (old yellow enzyme). isoform 3 Energy 
17 6.1 85.9 YER091c MET6 38 2.32 Homocysteine methyltransferase Amino acid biosynthesis 
18 5.7 38.6 YPR127w YPR127w 36 2.29 Similarity to C-term, of N. tabacum auxin-induced protein Unknown 
19 5.4 41.7 YFL039c ACT1 21 2.28 Actin Cytoskeleton 
20 5.5 57.7 YJR010w MET3 14 2.17 Sulfate adenylyltransferase Amino acid biosynthesis 
21 – – NI NI NI −2.19 NI NI 
22 5.7 115.9 YLR249w YEF3 11 −2.3 Translation elongation factor eEF3 Protein biosynthesis 
23 5.3 54.4 YPL061w ALD6 17 −2.32 Aldehyde dehydrogenase, cytosolic Carbon metabolism 
24 – – NI NI NI −2.33 NI NI 
25 5.3 66.6 YDL229w SSB1 59 −2.83 Heat shock protein of HSP70 family Stress 
26 – – NI NI NI −2.89 NI NI 
27 – – NI NI NI −3.34 NI NI 
No. pMw (kDa) ORF name Gene or ORF name % Coveragea Factor Name or function Group functions 
5.8 49.1 YER043c SAH1 24 5.5 S-adenosyl-l-homocysteine hydrolase Amino acid biosynthesis 
69.8 YAL005c SSA1 43 4.96 Heat shock protein of HSP70 family, cytosolic Stress 
9.2 57.7 YPR002w PDH1∗ 43 3.77 Similarity to B. subtilis mmgE protein Carbon metabolism 
5.5 54.8 YJR121w ATP2∗ 23 3.53 F1F0-ATPase complex, F1 β subunit Respiration 
4.8 74.5 YJL034w KAR2 44 3.35 Nuclear fusion protein Stress 
5.5 39.6 YKL060c FBA1∗ 17 2.78 Fructose-bisphosphate aldolase Carbon metabolism 
5.8 61.5 YLR044c PDC1 26 2.65 Pyruvate decarboxylase, isozyme 1 Carbon metabolism 
– – NI NI NI 2.61 NI NI 
– – NI NI NI 2.55 NI NI 
10 6.2 36.8 YOL086c ADH1 26 2.53 Alcohol dehydrogenase I Carbon metabolism 
11 69.8 YAL005c SSA1 28 2.48 Heat shock protein of HSP70 family, cytosolic Stress 
12 6.2 53.5 YHR183w GND1 34 2.45 6-phosphogluconate dehydrogenase Carbon metabolism 
13 4.6 50.9 YFL037w TUB2 29 2.41 β-Tubulin Cytoskeleton 
14 – – NI NI NI 2.38 NI NI 
15 – – NI NI NI 2.35 NI NI 
16 5.4 44.9 YPL171c OYE3 11 2.35 NAPDH dehydrogenase (old yellow enzyme). isoform 3 Energy 
17 6.1 85.9 YER091c MET6 38 2.32 Homocysteine methyltransferase Amino acid biosynthesis 
18 5.7 38.6 YPR127w YPR127w 36 2.29 Similarity to C-term, of N. tabacum auxin-induced protein Unknown 
19 5.4 41.7 YFL039c ACT1 21 2.28 Actin Cytoskeleton 
20 5.5 57.7 YJR010w MET3 14 2.17 Sulfate adenylyltransferase Amino acid biosynthesis 
21 – – NI NI NI −2.19 NI NI 
22 5.7 115.9 YLR249w YEF3 11 −2.3 Translation elongation factor eEF3 Protein biosynthesis 
23 5.3 54.4 YPL061w ALD6 17 −2.32 Aldehyde dehydrogenase, cytosolic Carbon metabolism 
24 – – NI NI NI −2.33 NI NI 
25 5.3 66.6 YDL229w SSB1 59 −2.83 Heat shock protein of HSP70 family Stress 
26 – – NI NI NI −2.89 NI NI 
27 – – NI NI NI −3.34 NI NI 
a

Percentage of matched peptide covering the protein obtained after spot identification by mass spectrometry and MS-Fit questioning. The Mw and pI of each protein are those given in the MIPS catalogue. NI indicates that a spot was not identified by MALDI-TOF. These proteins are reported by their numbers in Fig. 5B.

First generation (Fig. 5A)

A comparison between the beginning (a) and the end (b) of the first generation shows that the most significant changes concerned proteins involved in carbohydrate metabolism, respiration, and amino acid and protein biosynthesis.

On one hand, 50 of the 85 differentially expressed proteins during the first fermentation were repressed (Fig. 5A; a and b; Table 2). Most glycolytic enzymes, proteins involved in acetyl-CoA formation, proteins of the tricarboxylic acid cycle, but also proteins involved in respiration were down regulated. The abundance of other proteins involved in amino acid, nucleotide, or lipid synthesis decreased. On the other hand, most of the proteins induced were fragments belonging to either carbon metabolism, or protein or amino acid biosynthesis pathways.

Most of these drastic changes in protein expression reveal an adaptive response to anaerobic conditions. This is not surprising as yeasts had been propagated in aerobic conditions. Indeed, the down-regulation of carbohydrate metabolism, and in particular the decreased expression of the acetyl-CoA pathway and some mitochondrial proteins, directly correlates with a switch from respiratory to fermentative metabolism. The main metabolic pathways affected during this switch are represented in Fig. 6.

6

Metabolic pathways affected by aerobic-to-anaerobic switch during the first generation. Protein names followed by an asterisk designate fragments.

6

Metabolic pathways affected by aerobic-to-anaerobic switch during the first generation. Protein names followed by an asterisk designate fragments.

Impact of aerobic-to-anaerobic switch on glycolysis and acetyl-CoA pathways

Glycolysis was only moderately affected, since the only significant change was the repression of Glk1p, Hxk1p, Hxk2p and Tdh3p. However, the switch from respiratory to fermentative metabolism was accompanied by an increase of glycolytic protein fragments. Trabalzini and coworkers [22] have reported that most of the protein fragments which appear after glucose exhaustion, in a wine strain grown on YPD, are specific isoforms of glycolytic enzymes. This suggests that intracellular proteolysis has an impact on the regulation of the abundance of these isoenzymes. Consequently, the proteolysis observed here could be related to the stress caused by the oxidative-to-fermentative switch. There was, however, a clear inhibition of acetyl-CoA metabolism, especially of the pyruvate dehydrogenase bypass (PDH bypass). Pyruvate is processed into acetyl-CoA via three pathways: the pyruvate dehydrogenase complex (PDH complex), and the cytosolic and the mitochondrial pyruvate dehydrogenase bypasses. The PDH complex was represented by Pdb1p (pyruvate dehydrogenase) on our gel but its decrease was not significant (−1.4-fold). In contrast, expression of Ald6p and Ald4p, involved respectively in cytoplasmic and mitochondrial PDH bypasses, was strongly inhibited.

Ald6p is thought to be involved in the production of acetate leading to acetyl-CoA synthesis. Acetyl-CoA is then supplied principally to biosynthetic pathways, in particular for lipid synthesis and in smaller amounts for the Krebs cycle [30]. Ald4p has recently been identified as a mitochondrial PDH bypass [31] leading to acetate production in mitochondria. It has already been shown that both cytosolic and mitochondrial aldehyde dehydrogenases are required for growth on ethanol [32], but only the former has a role in acetate formation during sugar fermentation [33]. Consequently, down-regulation of Ald6p and Ald4p during the first generation suggests that they play a role during aerobic propagation on saccharose, and probably are involved in adaptation to fermentation conditions. Indeed, a strong (about 6-fold) decline in Ald6p expression probably leads to a consistent reduction in acetyl-CoA supply for the Krebs cycle but also partially for lipid synthesis. The protein encoded by the ACH1 gene, which catalyses hydrolysis of acetyl CoA in the mitochondrial compartment [35], also displayed significant repression (about 6-fold) during the first generation. Interestingly, Ach1p and Ald6p are both subject to repression by the cAMP-signalling pathways via the stress-responsive cis element (STRE) [36]. The simultaneous repression of both proteins thus would suggest that Ach1p is also involved in oxidative metabolism during propagation. Summarising, slowdown and adaptation to fermentation conditions appear to involve a down-regulation of glycolytic flux and less active PDH bypasses. Fine regulation of the latter will have to be studied further to better understand their contribution to biosynthesis and energy metabolism.

Other pathways

Concerning stress-response proteins, variable expression was noted for six of the fifteen proteins compared quantitatively. Ssc1p and Hsp60p, both involved in mitochondrial protein folding, decreased whereas expression of other heat-shock proteins such as Ssa4p and Hsp26p, as well as Kar2p and Pnc1p, two proteins also related to stress-response, were strongly enhanced.

Other pathways were also modified during the first generation. For example, two proteins, Yhb1p and Hem13p, were affected by the change from oxidative to fermentative metabolism. YHB1 is a gene encoding a flavohemoprotein whose expression is higher in aerobic conditions [38]. In fact, the decrease in the level of Yhb1p during the first generation goes in line with the switch from aerobic to anaerobic metabolism. Hem13p, known to be involved in heme biosynthesis, presented enhanced expression, which correlates with previous observations indicating that oxygen inhibits the encoding gene [39]. Since there is a major slowdown of the respiratory system, decreased Krebs cycle activity (Fum1p, Cit2p, Kgd1p and Kgd2p) is not surprising, as confirmed by the down-regulation of Atp2p, Cor1p and Rip1p, involved in the mitochondrial respiratory chain. Consequently, the slowdown of the PDH bypass seems to have an impact on lipid synthesis and the Krebs cycle which corroborates with the general slowdown of biosynthetic pathways such as the synthesis of sulphur (Sam2p, Sam1p, Met6p, and Met17p) and branched (Ilv5p) amino acids as well as arginine (Arg1p).

Third generation (Fig. 5B)

The yeast was re-pitched in a second and then in a third subsequent generation. Two-dimensional proteome analysis was performed at the same steps as for the first generation (beginning T1/end T3). Results are reported in Table 3 and illustrated in Fig. 5B (a and b). The overall pattern shows much less variation than during the first generation. Quantitatively, the degree of variation in expression was much lower than in the first fermentation; maximum variability was 5-fold, compared with 10-fold for the first generation. Spots exhibiting variability corresponded essentially to proteins involved in carbon metabolism, methionine biosynthesis and stress-response pathways. Only 27 spots with at least 2-fold changes were found and 74% corresponded to enhanced expression. Proteins involved in methionine biosynthesis such as Sah1p, Met6p and Met3p, as well as those involved in carbon metabolism, are found in these 74%. But the increase of the Adh1p and Pdc1p cannot be considered as really enhanced since variable spots of these proteins were considered as minor spots. Concerning stress-response proteins, Ssb1p was down-regulated, whereas Ssa1p and Kar2p clearly presented enhanced expression.

Unsurprisingly, in contrast to the first generation, no protein variations related to the aerobic–anaerobic switch were observed during the third generation.

Comparisons between first and third generation: stress-response proteins

Changes in stress-responsive proteins between the end of the first generation (F3) and the beginning (T1) of the third generation, especially the evolution of stress proteins, were studied (Table 4).

4

Evolution of some stress proteins during the fermentation process

Protein First generation Comparison between F3/T1 stages Third generation 
SSA4 Increased No change* No change 
HSP26 Increased No change No change 
PNC1 Increased No change No change 
KAR2 Increased Decreased Increased 
HSP60 Decreased No change No change 
SSC1 Decreased No change No change 
SSA No change No change Increased 
SSB1 No change Increased No change 
Protein First generation Comparison between F3/T1 stages Third generation 
SSA4 Increased No change* No change 
HSP26 Increased No change No change 
PNC1 Increased No change No change 
KAR2 Increased Decreased Increased 
HSP60 Decreased No change No change 
SSC1 Decreased No change No change 
SSA No change No change Increased 
SSB1 No change Increased No change 

“Increased” and “Decreased” indicate at least 2-fold change. All variations were established by quantitative analysis, except for proteins designated by an asterisk (∗) indicating visual comparison.

The adaptation to anaerobic conditions was characterized by enhancement of Hsp26p, Ssa4p, Pnc1p and Kar2p during the first generation. In the third generation, the enhanced level was maintained for all of these proteins except for Kar2p which decreased. Hsp26p is a general stress-responsive protein known to be induced upon exposure to a range of stress factors, such as entry into stationary phase or diauxic shift [42], sporulation [43,44], ethanol stress [45], osmostress [46], weak-acid exposure [37], heat shock [45] and H2O2 exposure [47]. Ssa4p, a member of the Hsp70 protein family, is heat- and ethanol-inducible [45,48] and is involved in chaperone functions preventing aggregation and allowing refolding of stress-damaged proteins. Pnc1p is also classified in the stress-response protein family since it is induced by sorbic acid stress [37,49] and ethanol treatment [50]. Thus, Hsp26p, Ssap4 and Pnc1p are stress proteins induced by a variety of treatments. Their induction during the first generation suggests that the oxidative-to-fermentative switch can be also considered as an environmental stress. Moreover, these proteins are also constitutively expressed in subsequent generations, and so are probably important for maintaining the viability of cells encountering stressful fermentative conditions.

KAR2 also encodes a chaperone protein of the HSP70 family and is required for protein folding in the endoplasmic reticulum (ER). In our study, Kar2p seems to be related to the process of adapting to fermentation-related stresses, since its expression was highly induced in both the first and third generations, but to a lesser extent at the beginning of the third fermentation (T1) than at the end of the first fermentation (F3).

On the other hand, Ssc1p and Hsp60p, involved in subcellular mitochondrial chaperone-assisted folding [51], both exhibited decreased expression, but only during the first fermentation. The down-regulation of these proteins is directly related to the general slowdown of mitochondrial activity especially for protein biogenesis.

Two other proteins, Ssb1p and Ssa1p, which both belong to the HSP family, show variable expression. Ssb1p and Ssb2p are ribosome-associated proteins which play a role in the folding of nascent polypeptides emanating from ribosomes [52]. Ssa1 to Ssa4 proteins form an essential chaperone group: at least one of them must be present at high levels to perform their chaperoning activity and assure cell viability. Ssb1 expression increased between the end of the first generation and the beginning of the third, whereas Ssa1p expression increased during the third generation. The role of these two proteins could not be clearly identified in these conditions.

Conclusions

This study confirms the relatedness between S. cerevisae S288c and the ale-brewing strain. The first protein reference map of an ale-brewing strain established here will be very helpful for future investigations concerning these kinds of brewing yeasts, but it also allowed us to study variations in protein expression during production-scale fermentation.

The most pronounced changes in protein expression occur in the first generation, during the switch from oxidative to fermentative metabolism. Besides the oxidative effect, the yeast has to cope with considerable change in nutrient supply due to the switch from synthetic media to wort. Yeast propagation is performed in synthetic saccharose medium whereas the wort used in the fermentation process principally contains maltose as well as glucose, fructose and small amounts of maltotriose and saccharose as carbon sources. Nevertheless, no drastic protein changes directly related to these important changes in sugar source were observed. The most significant changes observed were probably indirectly linked to glucose repression mechanisms involved in the adaptation of cells entering fermentation metabolism. In any case, the main effect of this adverse environmental change appears to be a general slowdown of acetyl-CoA metabolism via the PDH bypass and mitochondrial proteins. This result also highlights the huge impact of yeast preculture on metabolic changes occurring in the first generation of fermentations. Moreover, the great adaptability of brewing strains to fermentation conditions is revealed in the third generation where very few stress proteins are affected.

The complexity of the production-scale fermentation with many adverse effects does not allow to clearly identify biomarkers for the prediction of optimal fermentation progress. Although this study allows us to identify that Hsp26p, Ssa4p and Pnc1p as essential during fermentation, they cannot be used to predict a bad progression of fermentation.

Yeast cells must cope with many different kinds of stress during the brewing fermentation process. As highlighted in this study, stresses on brewing yeast must be tested on an individual strain basis to obtain a better understanding of the impact of each kind of stress on cell physiology and behaviour.

Acknowledgements

The authors would like to thank Pr. Alain Van Dorsselaer and Dr. Jean-Marc Strub for MS protein identification. Dominique Kobi is supported by a CIFRE fellowship from ANRT (Association nationale de la recherche technique).

References

[1]
Riou
C.
Nicaud
J.M.
Barre
P.
Gaillardin
C.
(
1997
)
Stationary-phase gene expression in Saccharomyces cerevisiae during wine fermentation
.
Yeast
 
13
,
903
915
.
[2]
DeRisi
J.L.
Vishwanath
R.L.
Brown
P.O.
(
1997
)
Exploring the metabolic and genetic control of gene expression on a genomic scale
.
Science
 
278
,
680
686
.
[3]
Ter Linde
J.J.M.
Liang
H.
Davis
R.W.
Steensma
H.Y.
Van Dijken
J.P.
Pronk
J.T.
(
1999
)
Genome-wide transcriptional analysis of aerobic and anaerobic chemostat cultures of Saccharomyces cerevisiae
.
J. Bacteriol.
 
181
,
7409
7413
.
[4]
Cavalieri
D.
Townsend
J.P.
Hartl
L.
(
2000
)
Manifold anomalies in gene expression in a vineyard isolate of Saccharomyces cerevisiae revealed by DNA microarray analysis
.
PNAS
 
97
,
12369
12374
.
[5]
Backhus
L.
DeRisi
J.
Bisson
L.F.
(
2001
)
Functional genomic analysis of a commercial wine strain of Saccharomyces cerevisiae under differing nitrogen conditions
.
FEMS Yeast Res.
 
1
,
111
125
.
[6]
Kuhn
K.M.
DeRisi
J.L.
Brown
P.O.
Sarnow
P.
(
2001
)
Global and specific translational regulation in the genomic response of Saccharomyces cerevisiae to a rapid transfer from a fermentable to a nonfermentable carbon source
.
Mol. Cell Biol.
 
21
,
916
927
.
[7]
Brejning
J.
Jespersen
L.
(
2002
)
Protein expression during lag phase and growth initiation in Saccharomyces cerevisiae
.
Int. J. Food Microbiol.
 
75
,
27
38
.
[8]
Rossignol
T.
Dulau
L.
Julien
A.
Blondin
B.
(
2003
)
Genome-wide monitoring of wine yeast gene expression during alcoholic fermentation
.
Yeast
 
20
,
1369
1385
.
[9]
Olesen
K.
Felding
T.
Gjermansen
C.
Hansen
J.
(
2002
)
The dynamics of the Saccharomyces carlsbergensis brewing yeast transcriptome during a production-scale lager beer fermentation
.
FEMS Yeast Res.
 
2
,
563
573
.
[10]
James
T.C.
Campbell
S.
Donnelly
D.
Bond
U.
(
2003
)
Transcription profile of brewery yeast under fermentation conditions
.
J. Appl. Microbiol.
 
9
,
432
448
.
[11]
Hansen
J.
Kielland-Brandt
M.C.
(
1994
)
Saccharomyces carlsbergensis contains two functional MET2 alleles similar to homologues from S. cerevisiae and S. monacensis
.
Gene
 
140
,
33
40
.
[12]
Borsting
C.
Hummel
R.
Schultz
E.R.
Rose
T.M.
Pedersen
M.B.
Knudsen
J.
Kristiansen
K.
(
1997
)
Saccharomyces carlsbergensis contains two functional genes encoding the acyl-CoA binding protein, one similar to the ACB1 gene from S. cerevisiae and one identical to the ACB1 gene from S. monacensis
.
Yeast
 
13
,
1409
1421
.
[13]
Tamai
Y.
Momma
T.
Yoshimoto
H.
Kaneko
Y.
(
1998
)
Co-existence of two types of chromosome in the bottom fermenting yeast, Saccharomyces pastorianus
.
Yeast
 
14
,
923
933
.
[14]
Joubert
R.
Brignon
P.
Lehmann
C.
Monribot
C.
Gendre
F.
Boucherie
H.
(
2000
)
Two-dimensional gel analysis of the proteome of lager brewing yeasts
.
Yeast
 
16
,
511
522
.
[15]
Casaregola
S.
Nguyen
H.V.
Lapathitis
G.
Kotyk
A.
Gaillardin
C.
(
2001
)
Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization
.
Int. J. Syst. Evol. Microbiol.
 
51
,
1607
1618
.
[16]
Pedersen
M.B.
(
1986
)
DNA sequence polymorphisms in the genus Saccharomyces III. Restriction endonuclease fragment patterns of chromosomal regions in brewing and other yeast strains
.
Carlsberg Res. Commun.
 
51
,
163
183
.
[17]
Tornai-Lehoczki
J.
Dlauchy
D.
(
2000
)
Delimination of brewing yeast strains using different molecular techniques
.
Int. J. Food Microbiol.
 
62
,
37
45
.
[18]
Azumi
M.
Goto-Yamamoto
N.
(
2001
)
AFLP analysis of type strains and laboratory and industrial strains of Saccharomyces sensu stricto and its application to phenetic clustering
.
Yeast
 
18
,
1145
1154
.
[19]
Boucherie
H.
Sagliogliocco
F.
Joubert
R.
Maillet
I.
Labarre
J.
Perrot
M.
(
1996
)
Two-dimensional gel protein database of Saccharomyces cerevisiae
.
Electrophoresis
 
17
,
1683
1699
.
[20]
Joubert
R.
Strub
J.M.
Zugmeyer
S.
Kobi
D.
Carte
N.
Van Dorsselaer
A.
Boucherie
H.
Jaquet-Gutfreund
L.
(
2001
)
Identification by mass spectrometry of two-dimensional gel electrophoresis-separated proteins extracted from lager brewing yeast
.
Electrophoresis
 
22
,
2969
2982
.
[21]
Boucherie
H.
Dujardin
G.
Kermorgant
M.
Monribot
C.
Slonimski
P.
Perrot
M.
(
1995
)
Two-dimensional protein map of Saccharomyces cerevisiae: construction of a Gene-Protein Index
.
Yeast
 
11
,
601
613
.
[22]
Trabalzini
L.
Paffetti
A.
Scaloni
A.
Talamo
F.
Ferro
E.
Coratza
G.
Bovalini
L.
Lusini
P.
Martelli
P.
Santucci
A.
(
2003
)
Proteomic response to physiological fermentation stresses in a wild-type wine strain of Saccharomyces cerevisiae
.
Biochem. J.
 
370
,
35
46
.
[23]
Polevoda
B.
Norbeck
J.
Takakura
H.
Blomberg
A.
Sherman
F.
(
1999
)
Identification and specificities of N-terminal acetyltransferases from Saccharomyces cerevisisae
.
EMBO J.
 
18
,
6155
6168
.
[24]
Guy
G.R.
Philip
R.
Tan
Y.H.
(
1994
)
Analysis of cellular phosphoproteins by two-dimensional gel electrophoresis: Application for cell signaling in normal and cancer cells
.
Electrophoresis
 
15
,
417
440
.
[25]
Futcher
B.
Latter
G.I.
Monardo
P.
McLaughlin
C.S.
Garrels
J.I.
A sampling of the yeast proteome
.
Mol. Cell. Biol.
  (
1999
)
7357
7368
.
[26]
Steinberg
T.H.
Pretty On Top
K.
Berggren
K.N.
Kemper
C.
Jones
L.
Diwu
Z.
Haugland
R.P.
Patton
W.F.
(
2001
)
Rapid and simple nanogram detection of glycoproteins in polyacrylamide gels and on electroblots
.
Proteomics
 
1
,
841
855
.
[27]
Patton
W.F.
(
2002
)
Detection technologies in proteome analysis
.
J. Chromatogr. B
 
771
,
3
31
.
[28]
Larsen
M.R.
Larsen
P.M.
Fey
S.J.
Roepstorff
P.
(
2001
)
Characterization of differently processed forms of enolase 2 from Saccharomyces cerevisiae by two-dimensional gel electrophoresis and mass spectrometry
.
Electrophoresis
 
22
,
566
575
.
[29]
Rogowska-Wrzesinska
A.
Larsen
P.M.
Blomberg
A.
Görg
A.
(
2001
)
Comparison of the proteomes of three yeast wild-type strains: CEN.PK2, FY1679 and W303
.
Comp. Funct. Genom.
 
2
,
207
225
.
[30]
Pronk
J.T.
Wenzel
T.J.
Luttik
M.A.
Klaassen
C.C.
Scheffers
W.A.
Steensma
H.Y.
Van Dijken
J.P.
(
1994
)
Energetic aspects of glucose metabolism in a pyruvate-dehydrogenase-negative mutant of Saccharomyces cerevisiae
.
Microbiology
 
140
,
601
610
.
[31]
Boubekeur
S.
Bunoust
O.
Camougrant
N.
Castroviejo
M.
Rigoulet
M.
Guérin
B.
(
1999
)
A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae
.
J. Biol. Chem.
 
274
,
21044
21048
.
[32]
Tessier
W.D.
Meaden
P.G.
Dickinson
F.M.
Midgley
M.
(
1998
)
Identification and disruption of the gene encoding the K+-activated acetaldehyde dehydrogenase of Saccharomyces cerevisiae
.
FEMS Microbiol. Lett.
 
164
,
29
34
.
[33]
Remize
F.
Andrieu
E.
Dequin
S.
(
2000
)
Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisisae: role of the cytosolic Mg2+ and mitochondrial K+ acetaldehyde dehydrogenases Ald6p and Ald4p in acetate formation during alcoholic fermentation
.
Appl. Environ. Microbiol.
 
66
,
3151
3159
.
[34]
Wackerbauer
K.
Beckmann
M.
(
2003
)
Preservation of brewers yeast. Part I: What role does intracellular trehalose content play
.
Brauwelt
 
21
,
48
53
.
[35]
Buu
L.M.
Chen
Y.C.
Lee
F.J.S.
(
2003
)
Functional characterization and localization of acetyl-CoA hydrolase, Ach1p, in Saccharomyces cerevisiae
.
JBC Papers
 
278
,
17203
17209
.
[36]
Boy-Marcotte
E.
Perrot
M.
Bussereau
F.
Boucherie
H.
Jacquet
M.
(
1998
)
Msn2p and Msn4p control a large number of genes induced at the diauxic transition which are repressed by cyclic AMP in Saccharomyces cerevisiae
.
J. Bacteriol.
 
180
,
1044
1052
.
[37]
Nobel
H.
Lawrie
L.
Brul
S.
Klis
F.
Davis
M.
Alloush
H.
Coote
P.
(
2001
)
Parallel and comparative analysis of the proteome and transcriptome of sorbic acid-stressed Saccharomyces cerevisiae
.
Yeast
 
18
,
1413
1428
.
[38]
Liu
L.
Zeng
M.
Hausladen
A.
Heitman
J.
Stamler
J.S.
(
2000
)
Protection from nitrosative stress by yeast flavohemoglobin
.
PNAS
 
97
,
4672
4676
.
[39]
Zagorec
M.
Buhler
J.M.
Treich
I.
Keng
T.
Guarente
L.
Labbe-Bois
R.
(
1988
)
Isolation, sequence and regulation of the yeast HEM13 gene coding for coproporphyrinogen oxidase
.
J. Biol. Chem.
 
15
,
9718
9724
.
[40]
Brosnan
M.P.
Donnelly
D.
James
T.C.
Bond
U.
(
2000
)
The stress response is repressed during fermentation in brewery strains of yeast
.
J. Appl. Microbiol.
 
88
,
746
755
.
[41]
Dengis
P.B.
Rouxhet
P.G.
(
1997
)
Surface properties of top and bottom-fermenting yeast
.
Yeast
 
13
,
931
943
.
[42]
Puig
S.
Pérez-Ortin
J.E.
(
2000
)
Stress response and expression patterns in wine fermentations of yeast genes induced at the diauxic shift
.
Yeast
 
16
,
139
148
.
[43]
Kurtz
S.
Rossi
J.
Petko
L.
Lindquist
S.
(
1986
)
An ancient developmental induction: heat-shock proteins induced in sporulation and oogenesis
.
Science
 
4742
,
1154
1157
.
[44]
Petko
L.
Lindquist
S.
(
1988
)
Hsp26 is not required for growth at high temperatures, nor for thermotolerance, spore development, or germination
.
Cell
 
45
,
885
894
.
[45]
Piper
W.P.
Talreja
K.
Panaretou
B.
Moradas-Ferreira
P.
Byrne
K.
Praekelt
U.M.
Meacock
P.
Récnacq
M.
Boucherie
H.
(
1994
)
Induction of major heat-shock proteins of Saccharomyces cerevisiae, including plasma membrane Hsp30, by ethanol levels above a critical threshold
.
Microbiology
 
140
,
3031
3038
.
[46]
Blomberg
A.
(
1997
)
Osmoresponsive proteins and functional assessment strategie in Saccharomyces cerevisiae
.
Electrophoresis
 
8
,
1429
1440
.
[47]
Godon
C.
Lagniel
G.
Lee
J.
Buhler
J.M.
Kieffer
S.
Perrot
M.
Boucherie
H.
Toledano
M.B.
Labarre
J.
(
1998
)
The H2O2 stimulon in Saccharomyces cerevisiae
.
J. Biol. Chem.
 
34
,
22480
22489
.
[48]
Parsell
D.A.
Taulien
J.
Lindquist
S.
(
1993
)
The role of heat-shock proteins in thermotolerance
.
Philos. Trans. R Soc. Lond. B – Biological Sciences
 
1289
,
279
285
.
[49]
Moskvina
E.
Schuller
C.
Maurer
C.T.
Mager
W.H.
Ruis
H.
(
1998
)
A search in the genome of Saccharomyces cerevisiae via stress response elements
.
Yeast
 
14
,
1041
1050
.
[50]
Ghislain
M.
Talla
E.
François
J.M.
(
2002
)
Identification and functional analysis of the Saccharomyces cerevisiae nicotinamidase gene, PNC1
.
Yeast
 
19
,
215
224
.
[51]
Lill
R.
Nargang
F.E.
Neupert
W.
(
1996
)
Biogenesis of mitochondrial proteins
.
Curr. Opin. Cell. Biol.
 
8
,
505
512
.
[52]
James
P.
Pfund
C.
Craig
E.A.
(
1997
)
Functional specificity among Hsp70 molecular chaperones
.
Science
 
5298
,
387
389
.