Walker Graham AdvancedYeastCourse
Transcript of Walker Graham AdvancedYeastCourse
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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
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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.
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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
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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
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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)
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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
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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
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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)
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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
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GLYCOLYSISGLYCOLYSIS
Glucose
PYRUVATE
2ADP
2ATP
2NAD+
2NADH
Acetaldehyde + CO2
Ethanol
2NADH
2NAD+
Mg
Zn
MetalsMetals& &
yeast stressyeast stress
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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?
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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
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7
8
9
Mg loss fg/cell
0% 5% 10% 15% 20%
Ethanol. %v/v
1 hour4 hour24 hour
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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)
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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
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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 (
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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
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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
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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
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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
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0
1 10 100 10000
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48 hours
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1 10 100 1000FL1
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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)
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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
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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
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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
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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
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20
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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)
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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
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
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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
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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
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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
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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
Inge Russell, Graham Stewart and WBC
Thank you!