The role of aquaporins in the freeze toleranceof yeast cells: application in frozen dough

Patrick Van Dijck

Department of Molecular MicrobiologyVIB

Laboratory of Molecular Cell BiologyK.U.Leuven

Excellent University, Bratislava 6 May 2008

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Nutrient-induced signal transduction in the yeast Saccharomyces cerevisiaeand 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

The frozen dough process

flour water yeast salt

mixing

dividing

moulding

freezing

storage at -20°C (1 day to 6 months)

thawing, proofing and baking

Nutrient-inducedloss of stressresistance

Trehalase activation

Rapid drop in general stress resistance

Trehalose mobilizationGlycogen mobilization

Repression ofSTRE-controlled genes

GlucosecAMP

PKA

Induction of ribosomal genes

etc.

Fermentation-induced loss of stress resistance

Fermenting yeast: low stress resistance

GlucoseSucrose

Fermentation

Stress resistance

Baker’s yeast

Brewer’s yeast

- frozen doughs

- high-gravity brewing

General observation in nature

Metabolic activity Stress 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

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

In general :

I. Prevention of trehalose mobilisation

Trehalose content Stress resistance

Dry baker’s yeast : Trehalose = ± 15 - 20% of dry weight (± 1-1.5 M in cytosol)

Initiation of fermentation :

Trehalose

Time

Stressresistance

Time

Glucose Glucose

Guaranteed to have no significant activity loss during storage for 2 years at room temperature

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 + Glu6P Tre6P Trehalose

NTH1

2 Glucose

Trehalose-6-P synthase

Trehalose-6-P phosphatase

Trehalase (neutral)

(Van Dijck et al. 1995 AEM 61, 109-115)

0

2

4

6

8

10

12

-15 0 15 30 45 60 75 90 105

Tre

hal

ose

(%

of

dry

w)

Time (min )

0

10

20

30

40

50

60

70

80

90

-15 0 15 30 45 60 75 90 105

% S

urv

ival

(h

eat

sho

ck o

f 10

min

at

52°C

)GlucoseGlucose

0

100

-15 0 15 30 45 60 75 90 10

5

wild type

tps1∆

pTPS1

nth1∆

Time (min )

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

Stressresistance

Time

Glucose

wild type strain

fil mutant

Heat shock

lab strain (heat stress)

Isolation of fil mutants

Yeast cells + EMS

50 ml YPD

1stat. phase 500 µl

50 ml YPD90 min 30 °C30 min 52 °C

2

stat.. phase500 µl

50 ml YPD90 min 30 °C

3

YPD plates

100 µl

30’ 52 °C 30’ 54 °C 30’ 56 °C

4

5

fil1 mutant

partially inactivating point mutation in adenylate cyclase: Cyr1E1682K

High(er) stress resistance and high metabolic activity are not incompatible

0.01

0.1

1

10

100

0 30 60 90

Time (min)

fil1

wild type

Glucose

(Van Dijck et al. 2000 JMMB 2, 521-530)

0

2

4

6

8

10

0 5 10 15 20

Time (h)

% S

urv

ival

(h

eat

sho

ck o

f 30

min

at

52°C

)

Gro

wth

(O

D 6

00 n

m)

0

50

100

150

200

0 5 10 15 20

Time (h)

Glucose Glucose

Eth

ano

l (m

mo

l/m

g d

ry w

)

Stress resistance Growth Fermentation

Why is the fil1 mutant more stress tolerant

Microarray analysis

A number of differentially regulated genesof 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?

GlucoseGlucose

cAMP cAMP

PKA PKA

Trehalose mobilisationRepression of STRE regulated genes

Rapid drop of general STRESS RESISTANCE

Fermentation

Adenylate Cyclase

Cap CapCyr1

Growth

Tps1Tps1Tps1Tps1 Hsp104Hsp104Hsp104Hsp104 Msn2-4Msn2-4Msn2-4Msn2-4

The fil1 mutation is mapped to the catalytic domain of the adenylate cyclase gene resulting in partial inactivation of AC

0

10

20

30

40

50

60

70

80

90

100

0 30 60 90 120

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)

Sur

viva

l (%

)

Deletion of transcription factors Msn2 and Msn4 in the fil1 mutant does not result in complete loss of heat stress resistance

% s

urv

ival

aft

er 1

5’ a

t 51

°C

Time after the addition of glucose (min)

fil1

fil1 msn2 msn4

wild type

msn2 msn4

Because of the existence of compensation effects, (cfr HSP104 expression)it is necessary to compare the presence or absence ofthe 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 fil1MDJ3: W303-1A tps1 hxk2 msn2 msn4 hsp104

0’

10’

30’

60’

MDJ2 (fil1)

MDJ3

MDJ2

MDJ3

MDJ2

MDJ3

MDJ2

MDJ3

The fil1 mutation strongly increases the heat stress resistance of a strainthat lacks trehalose, Hsp104 and all of the stress-regulated

Msn2/4 regulated genes1. On plates after heat-shock at 56 °C

WT

MDJ2

MDJ3

YPD 1.4 M NaCl5 mM H2O2

0

20

40

60

80

100

0 30 60 90 120

Time after addition of glucose (min)

Su

rviv

al a

fter

30’

at

48 °

C (

%)

MDJ2: tps1 hxk2 msn2 msn4 hsp104 fil1 ade2MDJ3: tps1 hxk2 msn2 msn4 hsp104 ade2PVD32: prototrophic W303-1A

The fil1 mutation strongly increases the heat stress resistance of a strainthat lacks trehalose, Hsp104 and all of the stress-regulated

Msn2/4 regulated genes2. In liquid medium during the start of fermentation

()

()()

W303-1A

hsp12hsp26

fil

fil hsp12fil hsp26

Control cultures

45 min 56 oC

60 min 56 oC

W303-1A

hsp12hsp26

fil

fil hsp12fil hsp26

W303-1A

hsp12hsp26

fil

fil hsp12fil hsp26 Vianna, submitted

0.001

0.01

0.1

1

10

100

0 15 30 45 60 75 90 105

% s

urvi

val (

15 m

in a

t 52

°C)

Time after addition of glucose (min)

Hsp26 is very important for the high heat stresstolerance of the fil1 mutant

WT

fil1

fil1 hsp26

hsp26

fil1 hsp12hsp12

OTHER, UNKOWN fil1 TARGETS??OTHER, UNKOWN fil1 TARGETS??

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

Fil1 Wild type

1. Stationary phase cells• 8 differentially expressed genes • 3 confirmed, 2 not confirmed by NB, 3 undetectable

2. 30 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

YC

R06

1W

HE

M13

TIS

11/C

TH

2

TR

P1

SS

C3

YG

L06

9cY

GL

218w

YN

L17

9cY

JR11

4wY

JL21

1C

YJL

160C

VM

A4

HX

T6

YD

L02

3cY

NL

190W

IDH

2

LE

U2

YO

R04

1cS

NU

13Y

JR12

6CY

ER

024W

TR

P1

YP

L27

6W

AC

H1

0

1

2

3

0

10

20

30

40

overexpressed in fil1

overexpressedin W303-1A

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∆

0

10

20

30

40

50

60

70

80

90

100

0 20 40 60 80 100

fil1

fil1 srf3∆

fil1 srf5∆

fil1 srf6∆

fil1 srf1∆fil1 srf2∆

fil1 srf4∆

Time after addition of glucose (min)

% s

urvi

val a

fter

hea

t sh

ock

PROBLEM: ALL THESE GENES OVERLAP WITH OTHER GENES

Introduction of the fil1 point mutation in industrial baker’s yeast strains

Despite a lot of effort, NO success

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

Freeze resistance (1h -30 °C)

AT25 mutant seemed to be improved in general stress resistance

0

20

40

60

80

100

120

0 30 60 90 120

Time (min)

Su

rviv

al (%

)

0

20

40

60

80

100

120

0 30 60 90 120

Time (min)S

urv

ival (%

)

Heat resistance (15’ 49°C)

GlucoseGlucose

AT25 mutant

S47 parent

AT25 mutant

S47 parent

Better heat and freeze resistance

Teunissen et al., AEM 2002

Much lower proofing-time compared to S47 after deep-freezingof dough to a core temperature of -30 °C

70

80

90

100

110

120

130

140

0 20 40 60 80 100

Time of storage at -20°C (days)

Pro

of-

Tim

e (

min

)

S47

AT25

Teunissen et al., AEM 2002

IV. Genome-wide expression analysis of ‘fil’ mutants

AT25 mutant

AT25 mutant S47 parent

AT25 mutant S47 parent

stress-sensitive strainsstress-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

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 ?

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

laboratory scale yeast industrial pilot scale yeast

Relative glucose consumption after freezing (RGC)

1 day 4°C (IGC)

1 day -30°C (FGC)

0

2

4

6

8

10

12

14

16

18

AT25 AT25+ AQY2

AT25+ AQY1

AT25 AT25+ AQY2

AT25+ AQY1

mM

glu

cose

con

sum

ed in

2.5

hOverexpression AQY1 or AQY2 in AT25 improves freeze resistance

36% 71% 54% 20% 97% 83%

Same effect with overexpression of human aquaporin gene hAQP1

1000

number of daysOverexpression 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

% s

urv

ival

10 20 30 40 50 60 70 80 90 1001

0

10

AT25

AT25 + AQY2

Overexpression of aquaporins improves freeze tolerance of C. albicans and S. pombe

Overexpression of AQY1-1 and AQY2-1 enhances freeze tolerance of industrial strains.

Other commercially important characteristics not affected.

Case study (6).

wild typebaker’s yeast

(AT25)

GMbaker’s yeast

(AT25 + AQY2-1)

non-frozencontrol

non-frozencontrol

frozen frozen

Sci

enti

sts@

wor

k 20

06.

Tanghe et al., 2002.

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/TPI1p AT25/TPI1p AQY1-1 AT25/TPI1p AQY2-1

Gassing power.

400

500

600

700

800

900

1000

0 20 40 60 80 100 120 140

frozen storage duration (days)

gass

ing

pow

er (

ml i

n 2

h) Proofing time.

50

60

70

80

90

100

110

120

0 20 40 60 80 100 120 140

frozen storage duration (days)p

roof

ing

tim

e (m

in a

t 35

°C)

large doughs, core T° -30°C

Case study (7).

Tanghe et al., 2004.

AT25+vector AT25+pAQY2-1BY4743+vector BY4743+pAQY2-1

Aquaporin-mediated improvement of freeze tolerance is restricted to fast freezing conditions.

survival in cell suspensions

N2,lSPL

EtOH -30°CSPL

freezer -30°CSPL

N2,lDPL

EtOH -30°CDPL

freezer -30°CDPL

0

20

40

60

80

100

120

%R

GC

laboratory strain BG industrial strain BG

EtOH -30°C SPLfreezer -30°C SPL freezer -30°C DPL

EtOH -30°C DPL

-25

-20

-15

-10

-5

0

5

10

15

20

25

2 4 6 8 10

large, industrial dough

tem

per

atu

re (

°C)

time (minutes)

Temperature evolution during freezing.

1

10

100

1000

0 10 20 30 40 50 60 70 80 90 100

frozen storage duration (days)

Fast freezing (EtOH -30°C).

surv

ial (

% C

FU

)

AT25 AT25/AQY2-1

LAT25 LAT25/AQY2-1

Slow freezing (freezer -30°C).

10

100

1000

0 10 20 30 40 50 60 70 80 90 100

frozen storage duration (days)su

rvia

l (%

CF

U)

AT25 AT25/AQY2-1

LAT25 LAT25/AQY2-1

survival in small doughs

Aquaporin-mediated improvement of freeze tolerance is restricted to fast freezing conditions.

Hypothesis.

water permeabilitylimiting?

aquaporin overexpression advantageous ?

chemical gradient for free water = unstable situation

EC freezingIC supercooling

critical cooling ratedependent on cell type

- S/V ratio - water permeability

fast freezing

IC ice crystal formation

slow freezingwater outflow

damage to cell organels and plasma membrane

survival ↓ ↓ ↓

cellular dehydration

survival ↓

Only with rapid freezingSlow 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

FREEZING

Osmoticgradient

H2OH2O

Extracellular medium frozen - Intracellular medium not frozen

Less intracellular ice crystal formation

Lower drop in viability

Rapid freezing

Slow freezing

Osmoticgradient

H2OH2O

Higher expression of aquaporins allows faster efflux of water

MCB LABMEETING 2007 @ Houffalize

Dr. Sonia ColomboDr. Barbara LeymanDr. Matthias VerseleDr. An TangheCristina Roscoe Vianna

Prof. Johan Thevelein

AcknowledgmentsAcknowledgments

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

marker 1 marker 2 etc.

Chr. I

A.

marker 1 marker 2 etc.

Figure 1

B.

I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI

C.

1/13

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

5. Mutant 6. Mutant 8. Mutant3. Mutant

1. Wild type 7. Wild type4. Wild type2. Wild type

+

++

+

+

+

+

+

C.

XAMS(Artificially Marked Strain)

Strain bearing two mutationsA.

+

+

B.

+

+

Figure 3

3/13

+

+

First technology allowing simultaneous identification of genes involved in polygenic traits

Many novel advanced applications