Yeast mineral nutrition and stresstolerance

Graeme Walker

Scotland
www.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

Major 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, Mg Essential trace ions: Zn, Ca, Fe, Mn, Mo, Ni Toxic trace ions: Pb, Cd, Cr, Hg, Cu, Al etc.

Why do yeasts need metals?

Cell growth Enzyme activity Cell structure

Stress tolerance

Cell -cell interactions

Cell division

Cereal wort

Mg Ca

Zn

ETHANOL

Fermentation

YEAST

propagation

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

FERMENTING CELLS Reticular mitochondria in Mg-replete yeast cells (grown in mM Mg media)

RESPIRING CELLS Vesicular mitochondria in Mg-limited yeast cells (grown in M Mg media)

Note: both culture conditions were aerobic with 5% glucose

RESPIRING CELLS Mg replenished in Mg-limited cultures

FERMENTING CELLS

Pyruvate decarboxylase and yeast cell Mg

700 Specific PDC activity (mol.min/mg protein) 600 500 400 300 200 100 0 366 509 Cell Mg (fg/cell) 628

Proposed role of Mg in yeast respirofermentative metabolism

GLUCOSE PYRUVATE

High Mg

PDC
FERMENTATION (CO 2, Ethanol)

Low Mg PDH

RESPIRATION (CO 2, H 2O, Energy)

Basically,
Low Mg FERMENTATION High Mg RESPIRATION

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 Ca

Mg
Ca

Mg

SUGARS

ETHANOL

Industrys view

Yeasts view
Calcium

CALCIUM

Magnesium

MAGNESIUM

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

Is there sufficient bioavailable Mg 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 fermentations (distillers yeast)

Ethanol (%v/v)
9 8 7 6 5 4 3 2 1 0 0 5 15 20 25 40 54 Control (3500ppm) Mg-supp (3600ppm)

Fermentation time, hours

Yeast preconditioning?
(mineral-enriched yeast for fermentation) Mineral supplements at yeast propagation, or re-hydration

Malt grist

Water

Zn, Mg
Mashing Wort
PRECONDITIONED YEAST

Fermentation Conditioning BEER

Ethanol productivity of Mgpreconditioned yeast

25 20 15 ng Ethanol/cell 10 5 0 1day 2day 4day 5day Fermentation time Control Preconditioned

GLYCOLYSIS
Glucose
2ADP 2ATP 2NAD+ 2NADH

Mg Zn

PYRUVATE

Acetaldehyde + CO2
2NADH 2NAD+

Ethanol

Metals
& yeast stress

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Stress factors for industrial yeasts

Osmostress Oxidative stress Cold shock Nutrient starvation, anaerobiosis Cell aging Ethanol /CO2 Mechanical sheer, hydrostatic pressure

Heat shock Acids/pH shock Dehydration/rehydration

Some 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 ion homeostasis?

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Stressed yeast cells lose Mg & 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

9 8 7 6

Mg loss 5 fg/cell 4
3 2 1 0 0% 5% 10% 15% 20%

1 hour 4 hour 24 hour

Ethanol. %v/v

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-ionised water prior to the addition of ethanol at the concentration and times indicated. Mg was measured in culture supernatants by atomic absorption spectrophotometry.

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2.0 Zn cell content (fg/cell) 1.8 1.5 1.3 1.0 0.8 0.5 0.3 0.0 1 5 Tim e (h)
Control H2O Temperature 45 Ethanol 20% Ethanol 20%+ Temperature 45

100 90 80 70 60 50 40 30 20 10 0 1 5 Time (h) 24

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Control H2O

Viability (%)

Temperature 45

Ethanol 20%

Ethanol 20%+ Temperature 45

Stressed brewing yeast cells lose Zn, and viability

Yeast stress-protection by intra & extracellular Mg

Ethanol stress (wine yeast)
100 90 80 70 60 50 40 30 20 10 0 0 1 2 3 4 5

Temp stress (brewing yeast)

100 90 80 70 60 50 40 30 20 10 0 0 1 2 3 4 5

Cell viability (%)

Control cells Preconditioned cells

2mM Mg 50mM Mg

Ethanol (10%) exposure, h

Heat shock (45C) exposure, h

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Ethanol-stressed wine yeast

(not Mg-preconditioned)

Ethanol-stressed wine yeast

(Mg-preconditioned)

Anti-stress functions of Mg?

Cell membrane stabilisation in the face of environmental insults (temp, ethanol, ROS, heavy metals) Stabilisation at the level of Mg chargeneutralisation of membrane phospholipids Elevated cell Mg suppresses stress proteins Additional roles as an antioxidant

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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 (<0.1ppm) may cause stuck fermentations and affect beer quality Stress protectant
Interacts with lipids to control membrane fluidity Interacts with nucleic acids to prevent free radical damage

Cellular location of zinc in yeast?

Cytoplasm low concentration Sequestration metallothioneins, zinc-finger proteins Compartmentalisation cell wall, vacuole

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Zinc uptake and homeostasis in yeast

Zrt1 high affinity system Zrt2 low affinity system Fet4 also transports Fe and Cu

Zinc uptake by brewing yeast

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LAGER strain-25C
100 90 Zn cell associated (fg/cell) 80 70 60 50 40 30 20 10 0 0 2 4 6 8 10 12 14 16 18 20 22 24 Tim e (h) 0.6 0.54 0.48 0.42 0.36 0.3 0.24 0.18 0.12 0.06 0 Zn supernatant (ppml)

Lager brewing yeast Zinc wort content is almost strain lab zero after 1 hour conditions
LAGER strain-25C
100 90 80 Percentage Zn cell associated (%) 70 60 50 40 30 20 10 0 0 1 2 3 4 5 6 7 Tim e (h)

Cell

Supernatant

Zn is entirely accumulated into cells which redistribute the metal among growing yeast

Influence of temperature on Zn uptake

a)
Log cell number (cells/ml) 1e+8

25C
Zn cell content (fg/cell) Zn supernatant (ppm) 120 100 80 0.6 0.5 0.4 0.3 0.2 0.1 0.0

c)
Log cell number (cells/ml) 1e+8

8C
Zn cell content (fg/cell) 100 80 0.5 0.4 0.3 0.2 0.1 0.0 Zn supernatant (ppm) 120 0.6

1e+7

60 40 20

1e+7

60 40 20

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

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

Zinc is entirely accumulated into yeast cells Zinc wort content is almost zero first few hours Zinc uptake is slower at lower temperatures Zinc uptake is metabolism-dependent

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Localisation of zinc in brewing yeast cells

Flow cytometric analysis of cellular Zn in yeast cells using RhodoRhodo-Zn1

0

200
0

160
0

Cells grown in 5.0ppm Zn

0

120 80
0

4 hours
1 10 100 1000

40

C O U N T S

0 1

10

100

1000

100 80 60 40 20 0 1 10 100 1000

48 hours

FL1

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Zinc is translocated to 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)

600

Starved or non-viable cells

500 400 300 200 100 0 0 0.8 1.6 3.2 6.4 12.8 102.4

~80% of Zn is insoluble (cell wall)

Cell Zn (fg/cell)
Zn uptake (after 6h)

Zn concentration, ppm

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Zinc & yeast fermentative performance

(simulated brewery fermentations)

Hopped-malt wort
(1060 S.G.) Pre-aerated wort Temperature: 14C

Duration: 11 days
Lager yeast Variable Zn

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Zn & fermentation performance

1.060 Specific gravity 1.050 1.040 1.030 1.020 1.010 1.000

Specific gravity

Low zinc (0.05 ppm) delayed sugar utilization

0 48 96 144 192 264 Time (hours)
0 0.05 0.12 0.48 1.07 10.8 (Zn ppm)

7 6 Ethanol (%) 5 4 3 2 1 0 48 96
0

Ethanol

High zinc (14 ppm) slightly delayed sugar utilization but same after 14 days fermentation 0.4 ppm-1.0 ppm Zn resulted in fastest fermentation rates

144 Time (hours)

0.05 0.12 0.48 1.07

192
10.83 (Zn ppm)

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Pilot Plant fermentations

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

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Zinc uptake during pilot-scale fermentations

1000
160

Zinc cell content (fg/cell)

Zn cell content (fg/cell)

800

0 ppm 0.5 ppm 1 ppm 5 ppm 10 ppm

140 120 100 80 60 40 20

600

400

200

0 88 138 Time (h) 162 186

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

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

Time (h)

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

Zn-limitation studies (Kluyver lab TUDelft)

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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

Decreased process efficiency

Perturbation

Biomarker

Identify perturbation specific changes in transcript levels using microarrays

Process optimised

Adjustment

Quality control

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

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Genes regulated during Zn-limitation

Regardless of O2: UP: 93 DOWN: 40 Anaerobic: DOWN: 36 Aerobic: UP: 119 DOWN: 16

UP: 77

Microarray analyses some interesting Zn

Zn vs C regulated genes

ZRT1 Zn uptake KTR6 Cell wall

mannosylation FLO11 flocculation

ILV2,3 amino acids ADH1,3,6 fermentation SOD1,2 stress

GSY2 GAC1 glycogen GLC3 PGM1,2 trehalose TPS1,2,3

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Biosynthesis of valine and isoleucine in S. cerevisiae

Pyruvate Pyruvate Acetolactate synthase ILV6 / ILV2 2-acetolactate 2-aceto-hydroxy butanoate Keto-acid reductoisomerase ILV5 2,3 dihydroxy 3-methyl 2,3 dihydroxy 3-methyl butanoate pentanoate Dihydroxyacid dehydratase 2-oxobutanoate

ILV3
2-oxoisovalerate 3-methyl 2-oxopentanoate Branched-chain amino acid transaminase Valine BAT1 / BAT2 Valyl-tRNA synthetase VAS1 Isoleucyl-tRNA synthetase ILS1 Isoleucine

Val-tRNA

Ile-tRNA

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

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Roles of Zn in brewing yeast key points

Zinc uptake is very rapid and complete Zinc is stored in the yeast vacuole and distributes to daughter cells at cell division 0.4 1.0 ppm Zinc appears optimal for active fermentation but may need more for respiration Zinc can influence beer flavour profile ZincZinc-limitation reveals new information on gene expression and potential influences on brewing fermentations

Summary
Metal ion bioavailability is important for yeast physiology & biotechnology (especially Mg, Zn) High Ca levels are detrimental Zinc uptake is very rapid and complete Cell Mg stimulates PDC and cell Zn stimulates ADH (and other genes, assessed by transcriptome profiles) MetalMetal-preconditioned yeasts are stressstress-tolerant and improve fermentation

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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?

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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

Thank you!

Inge Russell, Graham Stewart and WBC

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