1Graeme Walker

Yeast mineral nutrition and stressYeast mineral nutrition and stress--tolerancetolerance

Scotlandwww.abertay.ac.uk

WBC Advanced Yeast Workshop, Honolulu, Hawaii, August 2008

Outline

Metals & yeast physiology Metals and yeast stress-tolerance Mg, Ca & Zn in brewing fermentations Zinc & yeast transcriptomics Conclusions: industrial implications

2Major factors which impact on yeast fermentation performance

Yeast strain (genotype) Nutrients (including metal ions) Inhibitors (organic acids etc) Stress conditions (temp, ethanol, osmotic) Competitive microbes (bacteria, wild

yeasts)

Mineral nutrition of brewing yeast

Essential bulk minerals: P, S, K, MgEssential trace ions: Zn, Ca, Fe, Mn, Mo, NiToxic trace ions: Pb, Cd, Cr, Hg, Cu, Al etc.

3Why do yeasts need metals?

Cell structure

Cell -cellinteractions

Enzymeactivity

Stresstolerance

Cell division

Cell growth

Cereal wort

ETHANOL

Mg Zn? ?

YEAST

Ca

Fermentat ion

pr opaga ti on

4What about Mg (& Ca)?What about Mg (& Ca)?

Absolutely essential for yeast growth, cell cycle, metabolism and gene expression - Mg is the most abundant divalent cation in living cells!

Very high growth demand for Mg by yeast (Ks ~50M), supply (bioavailability) often not sufficient to meet demand

Stimulates yeast glycolysis and fermentative performance

Maintains yeast cellular structural integrity - stress protectant

Reticular mitochondriain Mg-replete yeast cells(grown in mM Mg media)

Vesicular mitochondriain Mg-limited yeast cells(grown in M Mg media)

FERMENTING CELLS RESPIRING CELLS

Note: both culture conditions were aerobic with 5% glucose

5Mg replenished in Mg-limitedcultures

RESPIRING CELLS

FERMENTING CELLS

Pyruvate decarboxylase and yeast cell Mg

0

100

200

300

400

500

600

700

366 509 628

Cell Mg (fg/cell)

Spec

ific

PDC

act

ivity

(

mol

.min

/mg

prot

ein)

6Proposed role of Mg in yeast respiro-fermentative metabolism

GLUCOSE

PYRUVATE

PDC PDH

FERMENTATION RESPIRATION(CO2, Ethanol) (CO2, H2O, Energy)

High Mg Low Mg

Basically,

FERMENTATION RESPIRATIONLow Mg

High Mg

7Magnesium v. Calcium?Magnesium v. Calcium? Cells actively include Mg,

but exclude Ca High growth demand for Mg,

but not for Ca Mg required for many enzymes,

Ca very few Ca antagonises Mg uptake and Mg-dependent

functions

SUGARS ETHANOL

MgCa

MgCa

IndustryIndustrys views view YeastYeasts views view

CALCIUM

Magnesium

Calcium

MAGNESIUM

Therefore: Maintain a high Mg:Ca ratio in fermentation media

8Is there sufficient bioavailableMg for optimal fermentation?

Yeast demand for Mg during fermentation is high

Mg bioavailability may not be sufficient to meet yeast demand

Increasing free Mg (and Mg:Ca) stimulates fermentation of Molasses Malt wort Wine must Cheese whey

Effect of Mg on molasses Effect of Mg on molasses fermentations (distillers yeast)fermentations (distillers yeast)

0123456789

0 5 15 20 25 40 54

Control (3500ppm)Mg-supp (3600ppm)

Fermentation time, hours

Ethanol (%v/v)

9Yeast preconditioning?(mineral-enriched yeast for fermentation)

Maltgrist Water

Mashing

Wort

Fermentation

Conditioning

BEER

Mineral supplements atyeast propagation, orre-hydration

Zn, Mg

PRECONDITIONEDYEAST

Ethanol productivity of Mg-preconditioned yeast

0

5

10

15

20

25

ng Ethanol/cell

1day 2day 4day 5dayFermentation time

ControlPreconditioned

10

GLYCOLYSISGLYCOLYSIS

Glucose

PYRUVATE

2ADP

2ATP

2NAD+

2NADH

Acetaldehyde + CO2

Ethanol

2NADH

2NAD+

Mg

Zn

MetalsMetals& &

yeast stressyeast stress

11

Stress factors for industrial yeastsEthanol

Dehydration/rehydration

Heat shockNutrientstarvation,anaerobiosis

/CO2

Acids/pH shockCell aging

Osmostress Mechanical sheer,hydrostatic pressure

Cold shockOxidative stress

Some Yeast Stress ResponsesSome Yeast Stress Responses Metabolism/production of trehalose &

glycerol Induction of heat/cold shock proteins Stress enzyme induction Cell membrane structural changes

Alteration of cellular metal ionhomeostasis?

12

Stressed yeast cells lose Stressed yeast cells lose Mg & ZnMg & Zn

Heat-shocked cells Ethanol-shocked cells

But, metal-enriched (eg. Mg-preconditioned) cells maintain viability under conditions imposed by heat-shock or toxic ethanol

Loss of Mg ions in ethanol-stressed brewing yeast.Cells (brewing strain of S. cerevisiae) were grown in malt broth, harvested, washed and re-suspended in de-ionisedwater prior to the addition of ethanol at the concentration and times indicated. Mg was measured in culturesupernatants by atomic absorption spectrophotometry.

0

1

2

3

4

5

6

7

8

9

Mg loss fg/cell

0% 5% 10% 15% 20%

Ethanol. %v/v

1 hour4 hour24 hour

13

0.0

0.3

0.5

0.8

1.0

1.3

1.5

1.8

2.0

1 5 24

Time (h)

Zn c

ell c

onte

nt (f

g/ce

ll)

Control H2O Temperature 45 Ethanol 20% Ethanol 20% + Temperature 45

0102030405060708090

100

1 5 24

Time (h)

Viab

ility

(%)

Control H2O Temperature 45 Ethanol 20% Ethanol 20% + Temperature 45

Stressed brewing yeast cells lose Zn, and viability

Yeast stressYeast stress--protection by protection by intra & intra & extracellularextracellular MgMg

0102030405060708090

100

0 1 2 3 4 5

Cel

l via

bilit

y (%

)

Control cellsPreconditioned cells

Ethanol stress (wine yeast)

0102030405060708090

100

0 1 2 3 4 5

2mM Mg50mM Mg

Ethanol (10%) exposure, h Heat shock (45C) exposure, h

Temp stress (brewing yeast)

14

Ethanol-stressed wine yeast(not Mg-preconditioned)

Ethanol-stressed wine yeast(Mg-preconditioned)

AntiAnti--stress functions of Mg?stress functions of Mg?

Cell membrane stabilisation in the face of environmental insults (temp, ethanol, ROS, heavy metals)

Stabilisation at the level of Mg charge-neutralisation of membrane phospholipids

Elevated cell Mg suppresses stress proteins Additional roles as an antioxidant

15

What about Zinc?What about Zinc? Essential trace element - rapidly taken up by yeast Needed for ~3% of yeast proteome function Essential for many enzymes e.g. ADH Zinc-limitation (

16

Zinc uptake and homeostasis in yeast

Zrt1 high affinity systemZrt2 low affinity systemFet4 also transports Fe and Cu

Zinc uptake by Zinc uptake by brewing yeastbrewing yeast

17

Zinc wort content is almost zero after 1 hour

Zn is entirely accumulated into cells which redistribute the metalamong growing yeast

LAGER strain-25C

0

10

20

30

40

50

60

70

80

90

100

0 2 4 6 8 10 12 14 16 18 20 22 24

Time (h)

Zn c

ell a

ssoc

iate

d (f

g/ce

ll)

0

0.06

0.12

0.18

0.24

0.3

0.36

0.42

0.48

0.54

0.6

Zn s

uper

nata

nt (p

pml)

Cell Supernatant

LAGER strain-25C

0

10

20

30

40

50

60

70

80

90

100

0 1 2 3 4 5 6 7

Time (h)

Perc

enta

ge Z

n ce

ll as

soci

ated

(%)

Lager brewing yeast strain labconditions

Zinc wort content is almost zero first few hours Zinc is entirely accumulated into yeast cells

Influence of temperature on Zn uptake

a)

Time (h)0 1 2 3 4 5 6 7

Log

cell

num

ber

(cel

ls/m

l)

1e+6

1e+7

1e+8

Zn c

ell c

onte

nt (f

g/ce

ll)

0

20

40

60

80

100

120

Zn su

pern

atan

t (pp

m)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

c)

Time (h)0 1 2 3 4 5 6 7

Log

cell

num

ber

(cel

ls/m

l)

1e+6

1e+7

1e+8

Zn

cell

cont

ent (

fg/c

ell)

0

20

40

60

80

100

120

Zn su

pern

atan

t (pp

m)

0.0

0.1

0.2

0.3

0.4

0.5

0.625C 8C

Zinc uptake is slower at lower temperatures Zinc uptake is metabolism-dependent

18

LocaliLocalissation of ation of zinc in brewingzinc in brewing

yeast cellsyeast cells

Cells grown in 5.0ppm Zn

4 hours

COUNTS

Flow cytometric analysis of cellular Zn in yeast cells using RhodoRhodo--Zn1Zn1

0

40

80

120

160

200

1 10 100 10000

0

0

0

0

0

1 10 100 10000

0

0

0

0

0

48 hours

101 100 1000

0

20

40

60

80

100

1 10 100 1000FL1

19

Zinc is Zinc is translocatedtranslocated to the vacuoleto the vacuole

Phase contrast(unstained cells)

Cells stained with Fluo-zinc 3

Cells stained with Cell-tracker B

Compartmentalisation of Zn in yeast

Actively-dividing, viable cells 60-90% of Zn is soluble(vacuolar)

Starved or non-viable cells ~80% of Zn is insoluble(cell wall)

0

100

200

300

400

500

600

0 0.8 1.6 3.2 6.4 12.8 102.4

Zn uptake (after 6h)

Cell Zn (fg/cell)

Zn concentration, ppm

20

Zinc & yeastZinc & yeastfermentative performance fermentative performance

(simulated brewery fermentations)(simulated brewery fermentations)

Hopped-malt wort(1060 S.G.) Pre-aerated wort Temperature: 14C Duration: 11 days Lager yeast Variable Zn

21

Zn & fermentation performance

Low zinc (0.05 ppm) delayedsugar utilization

High zinc (14 ppm) slightlydelayed sugar utilization but same after 14 days fermentation

0.4 ppm-1.0 ppm Zn resulted in fastest fermentation rates

1.000

1.010

1.020

1.030

1.040

1.050

1.060

0 48 96 144 192 264

Time (hours)

Spec

ific

grav

ity

0 0.05 0.12 0.48 1.07 10.8 (Zn ppm)

0

1

2

3

4

5

6

7

48 96 144 192 264Time (hours)

Eth

anol

(%)

0 0.05 0.12 0.48 1.07 10.83 (Zn ppm)

Specific gravity

Ethanol

Pilot Plant fermentations

Hopped-malt wort 14 Plato 200L, 11C Lager yeast Duration 8 days Variable Zn

22

Zn uptake patterns are similar to lab fermentations Sedimented yeast contains lower intracellular zinc concentration than yeast in suspension in the 0.5ppm fermentation.

Time (h)0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192

Zin

c ce

ll co

nten

t (fg

/cel

l)

0

200

400

600

800

1000

0 ppm 0.5 ppm 1 ppm 5 ppm 10 ppm

Zinc uptake during pilot-scale fermentations

0

20

40

60

80

100

120

140

160

88 138 162 186

Time (h)

Zn c

ell c

onte

nt (f

g/ce

ll)

0.5 ppm (susp.cells) 0.5 ppm (crop cells) 5 ppm (susp.cells) 5 ppm (crop cells)

Zn-limitation studies (Kluyver lab TUDelft)

23

Transcriptional responses of Saccharomyes cerevisiae

in zinc-limited chemostat culture

For the first time, we report operation of Zn-limited chemostats.

Genome-wide transcriptional responses* of aerobic and anaerobic Zn-limited chemostat cultures were compared to those obtained under C and N limitation

*Using Affymetrix Genechip microarrays.

Microarrays as diagnostic tools for yeast fermentations

Biomarker

Decreased process efficiency

Process optimised

Perturbation

Adjustment Quality control

Identify perturbationspecific changes in transcript levels using microarrays

From: Siew Leng Tai (2007) PhD thesis, TU Delft

24

Regardless of O2:UP: 93 DOWN: 40

Anaerobic:UP: 77 DOWN: 36

Aerobic:UP: 119 DOWN: 16

Genes regulated during Zn-limitation

Microarray analyses some interesting Zn

regulated genesZn vs C

ZRT1 Zn uptakeKTR6 Cell wall

mannosylationFLO11 flocculation

ILV2,3 amino acidsADH1,3,6 fermentation

SOD1,2 stressGSY2 GAC1 glycogenGLC3

PGM1,2 trehalose TPS1,2,3

25

Pyruvate 2-oxobutanoate

2-acetolactate 2-aceto-hydroxy butanoate

2,3 dihydroxy 3-methyl butanoate

2,3 dihydroxy 3-methyl pentanoate

Pyruvate

2-oxoisovalerate 3-methyl 2-oxopentanoate

Valine Isoleucine

Val-tRNA Ile-tRNA

Acetolactate synthase

ILV6 / ILV2

Keto-acid reductoisomerase

ILV5

Dihydroxyaciddehydratase

ILV3

Branched-chain amino acid transaminase

BAT1 / BAT2Valyl-tRNAsynthetase

VAS1Isoleucyl-tRNA

synthetaseILS1

Biosynthesis of valine and isoleucine in S. cerevisiae

Genes indicated in green were significantly down-regulated in response to Zn-limitation

What does it all mean?

Zn-limited cells:-are more oxidative stress-sensitive-have less glycogen & trehalose-may produce altered fusel oil profiles

More Zn needed to sustain aerobic growth

26

Roles of Zn in brewing yeast key points

Zinc uptake is very rapid and completeZinc uptake is very rapid and complete Zinc is stored in the yeast vacuole and distributes to daughter Zinc is stored in the yeast vacuole and distributes to daughter

cells at cell divisioncells at cell division 0.4 0.4 1.0 1.0 ppmppm Zinc appears optimal for active fermentation Zinc appears optimal for active fermentation

but may need more for respiration but may need more for respiration Zinc can influence beer flavour profileZinc can influence beer flavour profile ZincZinc--limitation reveals new information on gene expression and limitation reveals new information on gene expression and

potential influences on brewing fermentationspotential influences on brewing fermentations

Summary Metal ion Metal ion bioavailabilitybioavailability is important for yeast physiology & is important for yeast physiology &

biotechnology (especially Mg, Zn)biotechnology (especially Mg, Zn)

High Ca levels are detrimentalHigh Ca levels are detrimental

Zinc uptake is very rapid and completeZinc uptake is very rapid and complete

Cell Mg stimulates PDC and cell Zn stimulates ADH Cell Mg stimulates PDC and cell Zn stimulates ADH (and other genes, assessed by (and other genes, assessed by transcriptometranscriptome profiles)profiles)

MetalMetal--preconditioned preconditioned yeasts are stressyeasts are stress--tolerant and improve tolerant and improve fermentation fermentation

27

Industrial implications?

Important to monitor/optimise Mg, Ca and Zn in wort for yeast

Mineral requirements for propagation differ from fermentation

Metal ion depletion (Mg, Zn) may impact adversely on- Yeast stress resistance Fermentation performance, beer flavour

Important questions What are the optimal mineral levels in wort for

fermentation? Are these levels of mineral also optimal for yeast

propagation? Do metals influence yeast viability, vitality and

stress tolerance? How can we rapidly assess intracellular mineral

contents of yeast? What impact does metal-regulation of gene

expression have on brewing yeast performance?

28

Acknowledgements

Abertay University Yeast Group(Raffaele De Nicola, Jane White, Stelios Logothetis, Irma Ochigava, Paola Bruno, Vladimir Erdelji, Magdalena Nasiadaka, Rowena Shek, Biju Yohannan, Jason Bennett)

Delft University of Technology(Lucie hazelwood, Erik De Hulster, Theo Knijnenburg, Marcel Reinders,

Jack Pronk, Jean-Marc Daran, Pascale Daran-Lapujade)

Mike Walsh

Inge Russell, Graham Stewart and WBC

Thank you!