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The role of aquaporins in the freeze tolerance of yeast cells: application in frozen dough Patrick Van Dijck Department of Molecular Microbiology VIB Laboratory.

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Presentation on theme: "The role of aquaporins in the freeze tolerance of yeast cells: application in frozen dough Patrick Van Dijck Department of Molecular Microbiology VIB Laboratory."— Presentation transcript:

1 The role of aquaporins in the freeze tolerance of yeast cells: application in frozen dough Patrick Van Dijck Department of Molecular Microbiology VIB Laboratory of Molecular Cell Biology K.U.Leuven Excellent University, Bratislava 6 May 2008

2 VIB: Mission & Objectives mission –to conduct world class biomolecular research for the benefit of scientific progress and society objectives –strategic basic research –translate research results into products –public outreach & education

3 VIB’s Road to Success 1000 scientists and technicians 65 research groups in 8 departments Ghent Antwerp Louvain Brussels

4 Nutrient-induced signal transduction in the yeast Saccharomyces cerevisiae and the pathogens Candida glabrata and Candida albicans Biotechnological applications Fundamental research Red biotechnology Green biotechnology White biotechnology - stress resistance (baker’s, brewer’s, wine) - fermentation capacity (baker’s, brewer’s) - flavour ester synthesis (brewer’s) - bioethanol production - intestinal glucose sensing - antifungal targets - human diseases - trehalose metabolism - sugar sensing

5 The frozen dough process flour wateryeast salt mixing dividing moulding freezing storage at -20°C (1 day to 6 months) thawing, proofing and baking Nutrient-induced loss of stress resistance

6 Trehalase activation Rapid drop in general stress resistance Trehalose mobilization Glycogen mobilization Repression of STRE-controlled genes Glucose cAMP PKA Induction of ribosomal genes etc. Fermentation-induced loss of stress resistance

7 Fermenting yeast: low stress resistance Glucose Sucrose Fermentation Stress resistance Baker’s yeast Brewer’s yeast - frozen doughs - high-gravity brewing General observation in nature Metabolic activityStress resistance Industrial applications General question in biotechnology Is high metabolic activity compatible with high stress resistance ? To what extent can stress resistance of living cells be enhanced without compromising metabolic activity ? Initiation of fermentation

8 Development of yeast strains that maintain a high(er) stress resistance during active fermentation Stress response mechanisms: extensive information GOAL Stress resistance determinants: little information Improvement of stress resistance: very little information

9 In general : I. Prevention of trehalose mobilisation Trehalose content Stress resistance Dry baker’s yeast : Trehalose = ± % of dry weight (± M in cytosol) Initiation of fermentation : Trehalose Time Stress resistance Time Glucose Guaranteed to have no significant activity loss during storage for 2 years at room temperature

10 High trehalose levels cannot prevent loss of stress resistance in their absence Glucose causes disappearance of other factors required for stress resistance TPS1 TPS2 UDP-Glucose + Glu6PTre6PTrehalose NTH1 2 Glucose Trehalose-6-P synthase Trehalose-6-P phosphatase Trehalase (neutral) (Van Dijck et al AEM 61, ) Trehalose (% of dry w) Time (min ) % Survival (heat shock of 10 min at 52°C) Glucose Time (min )

11 II. Isolation of ‘fil’ mutants ‘fil’ mutants: deficient in fermentation-induced loss of stress resistance Procedure: EMS-mutagenesis / growth to stationary phase / fermentation for 90 min / (sub)lethal stress treatment (e.g. 30 min at 52°C) / repeat 1 more time and isolation of surviving mutants Stress resistance Time Glucose wild type strain fil mutant Heat shock lab strain (heat stress)

12 Isolation of fil mutants Yeast cells + EMS 50 ml YPD 1 stat. phase 500 µl 50 ml YPD 90 min 30 °C 30 min 52 °C 2 stat.. phase 500 µl 50 ml YPD 90 min 30 °C 3 YPD plates 100 µl 30’ 52 °C30’ 54 °C30’ 56 °C 4 5

13 fil1 mutant partially inactivating point mutation in adenylate cyclase: Cyr1 E1682K High(er) stress resistance and high metabolic activity are not incompatible

14 Why is the fil1 mutant more stress tolerant Microarray analysis A number of differentially regulated genes of which 6 are involved in the higher stress tolerance of the fil1 mutant Effect on expression of known targets of the general stress response pathway?

15 Glucose cAMP PKA Trehalose mobilisation Repression of STRE regulated genes Rapid drop of general STRESS RESISTANCE Fermentation Adenylate Cyclase Cap Cyr1 Growth Tps1Tps1Hsp104Hsp104Msn2-4Msn2-4 The fil1 mutation is mapped to the catalytic domain of the adenylate cyclase gene resulting in partial inactivation of AC

16 fil1 hxk2 tps1 hxk2 tps1 fil1 hxk2 hsp104 hxk2 hsp104 The presence of the fil1 mutation enhances heat stress resistance (20’ 51 °C) in strains that lack trehalose or Hsp104 Time after addition of glucose (min) Survival (%)

17 Deletion of transcription factors Msn2 and Msn4 in the fil1 mutant does not result in complete loss of heat stress resistance % survival after 15’ at 51 °C Time after the addition of glucose (min) fil1 fil1 msn2 msn4 wild type msn2 msn4

18 Because of the existence of compensation effects, (cfr HSP104 expression) it is necessary to compare the presence or absence of the fil1 mutation on the heat stress resistance in a strain that completely lacks trehalose, Hsp104 and the Msn2 and Msn4 transcription factors Construction of MDJ2: W303-1A tps1 hxk2 msn2 msn4 hsp104 fil1 MDJ3: W303-1A tps1 hxk2 msn2 msn4 hsp104

19 0’ 10’ 30’ 60’ MDJ2 (fil1) MDJ3 MDJ2 MDJ3 MDJ2 MDJ3 MDJ2 MDJ3 The fil1 mutation strongly increases the heat stress resistance of a strain that lacks trehalose, Hsp104 and all of the stress-regulated Msn2/4 regulated genes 1. On plates after heat-shock at 56 °C WT MDJ2 MDJ3 YPD1.4 M NaCl5 mM H 2 O 2

20 Time after addition of glucose (min) Survival after 30’ at 48 °C (%) MDJ2: tps1 hxk2 msn2 msn4 hsp104 fil1 ade2 MDJ3: tps1 hxk2 msn2 msn4 hsp104 ade2 PVD32: prototrophic W303-1A The fil1 mutation strongly increases the heat stress resistance of a strain that lacks trehalose, Hsp104 and all of the stress-regulated Msn2/4 regulated genes 2. In liquid medium during the start of fermentation ()() ()() ( )

21 W303-1A hsp12  hsp26  fil fil hsp12   fil hsp26   Control cultures 45 min 56 o C 60 min 56 o C W303-1A hsp12  hsp26  fil fil hsp12   fil hsp26   W303-1A hsp12  hsp26  fil fil hsp12   fil hsp26   Vianna, submitted % survival (15 min at 52 °C) Time after addition of glucose (min) Hsp26 is very important for the high heat stress tolerance of the fil1 mutant WT fil1 fil1 hsp26 hsp26 fil1 hsp12 hsp12 OTHER, UNKOWN fil1 TARGETS?? OTHER, UNKOWN fil1 TARGETS??

22 Micro-array analysis between fil1 and wild type (diauxic shift) Fil1 Wild type

23 1. Stationary phase cells 8 differentially expressed genes 3 confirmed, 2 not confirmed by NB, 3 undetectable min after addition of glucose to stationary phase cells 8 differentially expressed genes 6 confirmed by NB, 2 undetectable 3. During diauxic shift (glucose to ethanol shift) 31 differentially expressed genes (24 novel ORF’s) 20 confirmed, 3 not confirmed by NB, 8 undetectable 47 genes were selected after micro-array analysis 27 genes were confirmed by Northern blot analysis

24 YCR061W HEM13 TIS11/CTH2 TRP1 SSC3 YGL069c YGL218w YNL179c YJR114w YJL211C YJL160C VMA4 HXT6 YDL023c YNL190W IDH2 LEU2 YOR041c SNU13 YJR126C YER024W TRP1 YPL276W ACH overexpressed in fil1 overexpressed in W303-1A

25 Deletion of each SRF gene in the fil1 background results in loss of heat stress resistance Deletion of each SRF gene in the fil1 background results in loss of heat stress resistance fil1 56°C 0’ 30’ 60’ 120’ fil1 / srf3∆ fil1 / srf5∆ fil1 / srf2∆ fil1 / srf6∆ fil1 / srf1∆ fil1 / srf4∆ fil1 fil1 srf3∆ fil1 srf5∆ fil1 srf6∆ fil1 srf1∆ fil1 srf2∆ fil1 srf4∆ Time after addition of glucose (min) % survival after heat shock PROBLEM: ALL THESE GENES OVERLAP WITH OTHER GENES

26 Introduction of the fil1 point mutation in industrial baker’s yeast strains Despite a lot of effort, NO success

27 III. Isolation of ‘fil’ mutants Procedure: UV-mutagenesis / growth to stationary phase / preparation of small doughs (0.5g) / fermentation at 30°C for 30 min / freeze/thaw treatment up to 200 times (-30°C/20°C) / solubilization of dough / plating for survivors industrial strain Strain: commercial tetraploid/aneuploid strain S47 (Lesaffre, Lille) Purpose: freeze-resistant strain for use in frozen dough application - many stress-resistant mutants, but most with reduced growth and/or fermentation rate Results: - most promising mutant strain: AT25

28 Freeze resistance (1h -30 °C) AT25 mutant seemed to be improved in general stress resistance Time (min) Survival (%) Time (min) Survival (%) Heat resistance (15’ 49°C) Glucose AT25 mutant S47 parent AT25 mutant S47 parent Better heat and freeze resistance Teunissen et al., AEM 2002

29 Much lower proofing-time compared to S47 after deep-freezing of dough to a core temperature of -30 °C Time of storage at -20°C (days) Proof-Time (min) S47 AT25 Teunissen et al., AEM 2002

30 IV. Genome-wide expression analysis of ‘fil’ mutants AT25 mutant S47 parent AT25 mutantS47 parent stress-sensitive strains stress-resistant strains S47 sensitive derivativesAT25 resistant derivatives 3 genes consistently upregulated ≥ 3 times + 3 genes consistently downregulated ≤ 3 times in all resistant strains compared to all sensitive strains Confirmed by Northern Individual overexpression (in AT25) or individual deletion (lab strain): little effect

31 HOWEVER: AQY2 also overexpressed in some resistant strains Deletion and overexpression of AQY1 and AQY2 (and human hAQP1): effect on freeze tolerance ? AQY1 and AQY2 - two water channel encoding genes in yeast - inactive in many lab strains - deletion and overexpression: no clear phenotype - microbial aquaporins: function ?

32 stationary phase cells glucose glucose consumption (plate count) RGC (%) = (FGC/IGC)*100 = fermenting cells = non-fermenting cells no glucose 0°C -30°C IGC FGC Freeze tolerance assay. RGC (%) IGC FGC survival (%) = (CFU1/CFU2)*100 (CFU1) (CFU2) CFU (%) IGC FGC

33 laboratory scale yeastindustrial pilot scale yeast Relative glucose consumption after freezing (RGC) 1 day 4°C (IGC) 1 day -30°C (FGC) AT25 + AQY2 AT25 + AQY1 AT25 + AQY2 AT25 + AQY1 mM glucose consumed in 2.5h Overexpression AQY1 or AQY2 in AT25 improves freeze resistance 36% 71% 54%20%97%83% Same effect with overexpression of human aquaporin gene hAQP1

34 1000 number of days Overexpression of aquaporins improves maintenance of viability and fermentative activity during freeze storage Overexpression of aquaporin in AT25 improves maintenance of viability during freeze storage of small rapidly-frozen doughs AT25 (lab scale) AT25 + AQY2 (lab scale) AT25 (pilot scale) AT25 + AQY2 (pilot scale) 100 % survival AT25 AT25 + AQY2 Overexpression of aquaporins improves freeze tolerance of C. albicans and S. pombe

35 Overexpression of AQY1-1 and AQY2-1 enhances freeze tolerance of industrial strains. Other commercially important characteristics not affected. Case study (6). wild type baker’s yeast (AT25) GM baker’s yeast (AT25 + AQY2-1) non-frozen control non-frozen control frozen Tanghe et al., 2002.

36 Aquaporin overexpression does not improve yeast freeze tolerance when cultured and tested in industrial conditions. laboratory versus industrial conditions: many ≠ parameters - culturing conditions? NO - thawing conditions? NO - freezing conditions? YES AT25/TPI1pAT25/TPI1p AQY1-1AT25/TPI1p AQY2-1 Gassing power frozen storage duration (days) gassing power (ml in 2 h) Proofing time frozen storage duration (days) proofing time (min at 35°C) large doughs, core T° -30°C Case study (7). Tanghe et al., 2004.

37 AT25+vector AT25+pAQY2-1 BY4743+vector BY4743+pAQY2-1 Aquaporin-mediated improvement of freeze tolerance is restricted to fast freezing conditions. laboratory strain BGindustrial strain BG

38 EtOH -30°C SPL freezer -30°C SPL freezer -30°C DPL EtOH -30°C DPL Temperature evolution during freezing.

39 frozen storage duration (days) Fast freezing (EtOH -30°C). survial (% CFU) AT25 AT25/AQY2-1 LAT25 LAT25/AQY2-1 Slow freezing (freezer -30°C) frozen storage duration (days) survial (% CFU) AT25 AT25/AQY2-1 LAT25 LAT25/AQY2-1 survival in small doughs Aquaporin-mediated improvement of freeze tolerance is restricted to fast freezing conditions.

40 Hypothesis. water permeability limiting? aquaporin overexpression advantageous ? chemical gradient for free water = unstable situation EC freezing IC supercooling critical cooling rate dependent on cell type - S/V ratio - water permeability fast freezing IC ice crystal formation slow freezing water outflow damage to cell organels and plasma membrane survival ↓ ↓ ↓ cellular dehydration survival ↓

41 Only with rapid freezing Slow freezing: no effect (Larger commercial doughs: no effect unfortunately) Aquaporins play a function in freeze tolerance of yeast First clear function for microbial aquaporins Osmolarity low high FREEZING Underlying mechanism Extracellular medium freezes first Intracellular medium freezes later aquaporin

42 FREEZING Osmotic gradient H2OH2O H2OH2O Extracellular medium frozen - Intracellular medium not frozen Less intracellular ice crystal formation Lower drop in viability Rapid freezing Slow freezing Osmotic gradient H2OH2O H2OH2O Higher expression of aquaporins allows faster efflux of water

43 MCB LABMEETING Houffalize

44 Dr. Sonia Colombo Dr. Barbara Leyman Dr. Matthias Versele Dr. An Tanghe Cristina Roscoe Vianna Prof. Johan Thevelein Acknowledgments

45 Stress resistance in general is a multifactorial trait. Development of a novel technology to determine the all genes involved in a certain phenotype in one go. = AMTEM

46 Yeast strain with 600 specific, artificial oligonucleotide markers spread evenly throughout the genome

47 First technology allowing simultaneous identification of genes involved in polygenic traits Many novel advanced applications


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