The use of multiple deletion mutants for determining the physiological role of alcohol dehydrogenase isozymes in Saccharomyces cerevisiae James du Preez.

The use of multiple deletion mutants for determining the physiological role of alcohol dehydrogenase isozymes in Saccharomyces cerevisiae James du Preez *, Olga de Smidt, Laurinda Steyn & Koos Albertyn Department of Microbial, Biochemical & Food Biotechnology University of the Free State, Bloemfontein, South Africa

Cytosol glucose ethanol glucose glycerol ethanol Pyc Adh2 Adh1
glycolysis Pyc Adh2 Adh1 oxaloacetate pyruvate Pdc acetaldehyde Ald TCA cycle pyruvate acetate Pdh Acetyl-CoA Acs Cytosol amino acids acetyl-CoA acetyl-CoA acetate lipids acetaldehyde Mitochondrion Adh3 ? ethanol Pdh: pyruvate dehydrogenase complex Pyc: pyruvate carboxylase Pdc: pyruvate decarboxylase Ald: acetaldehyde dehydrogenase Acs: acetyl-coenzyme A synthetase Adh: alcohol dehydrogenase

Alcohol dehydrogenases in Saccharomyces cerevisiae
Three cytosolic alcohol dehydrogenases Acetaldehyde Ethanol Adh1 Adh2 NAD+ NADH Adh1 (fermentative) & Adh2 (oxidative): regarded as the two main enzymes involved in ethanol production and consumption, respectively Adh2: - Required for growth on ethanol - Repressed by glucose - Derepressed on non-fermentable carbon substrates, e.g. ethanol, glycerol Dickinson et al. (2003) J. Biol. Chem. 10: Adh5: - No distinct function. - No data on its kinetic characteristics, either in vitro or in vivo

Mitochondrial matrix alcohol dehydrogenase NAD+ NADH Adh3: involved in reversible ethanol acetaldehyde shuttle Adh1 / 2 Cytosol Maintenance of mitochondrial redox balance under anaerobic conditions: transfers redox equivalents from mitochondrial matrix to cytosol, where redox balance is restored by glycerol production. ethanol acetaldehyde Mitochondrial membranes Bakker et al. (2000) J. Bacteriol. 182: ethanol acetaldehyde Adh3 Mitochondrial matrix Suggested that aerobic ethanol oxidation may proceed also via the mitochondrial Adh3 NAD+ NADH Larsson et al. (1998) Yeast 14:

Adh4: - Controversy: mitochondrial or cytoplasmic? - Very low activity in laboratory strains - Metabolic role unknown Williamson & Paquin (1987) Mol. Gen. Genet. 209: Drewke & Ciriacy (1988) Biochim. et Biophys. Acta 950: 54-60 - Substantial activity in brewing yeast strains & some lab strains Dickinson et al. (2003) J. Biol. Chem. 10: - Can substitute for Adh1 in fermentative growth on glucose when over-expressed Paquin & Williamson (1986) Mol. Cell. Biol. 6: 70-79

Cultivation of S. cerevisiae W303-1A
Continuous culture Carbon-limited chemostat at 30°C, pH 5.5, DOT > 30% saturation, ml culture volume Chemically defined medium, with vitamins & amino acids. Ethanol Glucose Dcrit Batch cultivation Similar conditions, ml culture volume.

Chemostat steady-state culture pulsed with glucose (14 g l-1)
Genomic ADH expression levels in strain W303-1A Chemostat steady-state culture pulsed with glucose (14 g l-1) ADH1 ADH2 ADH3 Northern blot analyses Normalised ADH, % Biomass Ethanol Ethanol washout curve Acetic acid Glucose Time, min

Chemostat steady-state culture pulsed with ethanol (8 g l-1)
Genomic ADH expression levels in strain W303-1A Chemostat steady-state culture pulsed with ethanol (8 g l-1) ADH1 ADH2 ADH3 Northern blot analyses Normalised ADH, % Biomass Ethanol Ethanol washout curve Acetic acid Time, min

Engineered quadruple deletion mutants
Q1 ADH1 W303-1A, MATa, adh2::URA3, adh3::TRP1, adh4::HIS3, adh5::LEU2 Q2 ADH2 W303-1A, MATa, adh1::URA3, adh3::TRP1, adh4::HIS3, adh5:: LEU2 Q3 ADH3 W303-1A, MATa, adh1::TRP1, adh2::URA3, adh4::HIS3, adh5::LEU2 Q4 ADH4 W303-1A, MAT, adh1::HIS3, adh2::URA3, adh3::TRP1, adh5:: LEU2 Q5 ADH5 W303-1A, MATa, adh1::LEU2, adh2::URA3 adh3::TRP1, adh4::HIS3

Growth of S. cerevisiae ADH1 adh2 adh3 adh4 adh5 deletion mutant in glucose-limited chemostat culture Parental strain Parental strain Q1 Q1

Growth of S. cerevisiae ADH1 adh2 adh3 adh4 adh5 quadruple deletion mutant in batch culture on glucose Q1 Biomass Biomass Ethanol Ethanol Glucose Acetic acid Glucose Glycerol Glycerol Acetic acid Parental strain ADH1 quadruple deletion mutant

Growth of S. cerevisiae ADH2 adh1 adh3 adh4 adh5 quadruple deletion mutant in batch culture on glucose Q2 Biomass Glycerol Ethanol Biomass Acetaldehyde Glucose Glycerol Acetic acid Glucose Ethanol Parental strain ADH2 quadruple deletion mutant

Growth of S. cerevisiae ADH2 adh1 adh3 adh4 adh5 & ADH3 adh1 adh2 adh4 adh5 in batch culture on glucose Q2 Q3 Glycerol Glycerol Biomass Biomass Acetaldehyde Acetaldehyde Glucose Ethanol Glucose Ethanol ADH2 quadruple deletion mutant ADH3 quadruple deletion mutant

Cytosol glucose ethanol glucose glycerol ethanol Pyc Adh2 Pdc
glycolysis NADH NAD+ Pyc Adh2 NADH NAD+ oxaloacetate pyruvate Pdc acetaldehyde Ald TCA cycle pyruvate acetate Pdh Acetyl-CoA Acs Cytosol amino acids acetyl-CoA acetyl-CoA lipids Mitochondrion Pdh: pyruvate dehydrogenase complex Pyc: pyruvate carboxylase Pdc: pyruvate decarboxylase Ald: acetaldehyde dehydrogenase Acs: acetyl-coenzyme A synthetase Adh: alcohol dehydrogenase

Growth of S. cerevisiae ADH2 adh1 adh3 adh4 adh5 & ADH3 adh1 adh2 adh4 adh5 in batch culture
Carbon source Glucose Ethanol Parameter Parental strain Q2 Q3 Q1 & max, h-1 0.44 0.21 0.19 0.20 0.15 Yx/s 0.195 0.07 0.50 0.61 Yp/s, ethanol 0.38 0.13 0.11 Yp/s, glycerol 0.03 0.33 0.35 Qpmax, g l-1 h-1 0.86 0.05 0.06 Glycerol, g l-1 0.48 3.1 3.2 Acetic acid, g l-1 0.22 Acetaldehyde, g l-1 1.23 0.92

glycolysis Pyc Adh2 Adh1 oxaloacetate pyruvate Pdc acetaldehyde Ald TCA cycle pyruvate acetate Pdh Acetyl-CoA Acs Cytosol amino acids acetyl-CoA acetyl-CoA acetate lipids acetaldehyde Mitochondrion Adh3 ethanol ethanol Pdh: pyruvate dehydrogenase complex Pyc: pyruvate carboxylase Pdc: pyruvate decarboxylase Ald: acetaldehyde dehydrogenase Acs: acetyl-coenzyme A synthetase Adh: alcohol dehydrogenase

Growth of S. cerevisiae ADH4 adh1 adh2 adh3 adh5 and ADH5 adh1 adh2 adh3 adh4 on glucose
No growth on ethanol Weak growth on glucose Biomass Glucose Glycerol Ethanol Acetaldehyde Q4 Biomass Glucose Glycerol Ethanol Acetaldehyde Q5

glycolysis NADH NAD+ Pyc Adh2 Adh1 oxaloacetate pyruvate Pdc acetaldehyde Ald TCA cycle pyruvate acetate Pdh Acetyl-CoA Acs Cytosol amino acids acetyl-CoA acetyl-CoA lipids Mitochondrion Pdh: pyruvate dehydrogenase complex Pyc: pyruvate carboxylase Pdc: pyruvate decarboxylase Ald: acetaldehyde dehydrogenase Acs: acetyl-coenzyme A synthetase Adh: alcohol dehydrogenase

ADH transcription levels monitored by real-time RT-PCR analyses: glucose
Ethanol Q5 Q4 Q2 Q1 Q3 Log ADH, copies  l-1 Log ADH, copies l-1 Deletion mutants Parental strain, W303-1A Glucose, g l-1 Ethanol, g l-1 Glucose Ethanol

ADH transcription levels monitored by real-time RT-PCR analyses: ethanol
Deletion mutant Q2 Deletion mutant Q1 ADH1 ADH3 Deletion mutant Q3 Deletion mutants ADH1 ADH2 ADH3 ADH1 Parental strain, W303-1A ADH2 ADH3 ADH5

Regulation of ADH2 in chemostat and batch culture
Ethanol regulation of W303-1A ADH2 differed in chemostat and batch cultures Ethanol Chemostat response to glucose pulse Batch culture on glucose Normalised ADH, % Log ADH, copies l-1 Ethanol, g l-1 Ethanol, g l-1 Ethanol Batch culture on ethanol Chemostat response to ethanol pulse Normalised ADH, % Log ADH, copies l-1 Ethanol, g l-1 Ethanol, g l-1

Conclusions Adh1 the only alcohol dehydrogenase necessary for normal aerobic growth on glucose or ethanol; can substitute well for Adh2. Strains lacking ADH1 exhibited impaired aerobic growth and ethanol production on glucose with the formation of acetaldehyde and glycerol. Ethanol utilisation can proceed via the mitochondrial Adh3. Adh4 or Adh5 alone does not permit aerobic growth on ethanol. Adh4 or Adh5 apparently hardly capable of reducing acetaldehyde to ethanol: poor growth on glucose due to accumulation of NADH and acetaldehyde. Ethanol causes transcriptional repression of ADH2 in chemostat culture, but not in batch culture.

Acknowledgements Students Jacques Maré Olga de Smidt Laurinda Steyn
Co-author Koos Albertyn Supported by National Research Foundation, South Africa

Adh1, 2, 3, 5 High degree of homology between isozymes Adh1, 2, 3, 5 Adh1 with Adh2: 95% Adh1, 2, 3, 5: 78% Adh4 not homologous; significant kinetic similarity to Adh1

Regulation of ADH2 promoter in S. cerevisiae Y294 & CEN.PK 110-6C
Transformed with XYN2 from Trichoderma reesei using a promoter- terminator expression cassette derived from S. cerevisiae ADH2 gene. Episomal multicopy plasmid La Grange et al. (1996) Appl Environ. Microbiol. 62: Transcription of endo--(1,4)-xylanase gene (reporter gene) under control of ADH2 promoter. Continuous cultivation: Carbon-limited chemostat at 30°C, pH 5.5, DOT > 30% saturation, ml culture volume. Batch cultivation: similar conditions, ml culture volume.

Aerobic glucose-limited chemostat culture of S. cerevisiae W303-1A
Ethanol Glucose Dcrit Biomass

XYN2 mRNA ACT1 mRNA Time (h)
200 20 180 18 intracellular xylanase production, nkat/(mg.h) Specific rate of 160 16 Extracellular xylanase Activity (nkat/ml) 140 Intracellular xylanase Activity (nkat/ml) extracellular xylanase production, nkat/(mg.h) Specific rate of 14 120 12 100 10 80 8 60 6 40 4 20 2 -10 10 20 30 3 1.6 6 Biomass 1.4 2.5 5 1.2 2 4 1 Ethanol (g/l) Glucose (g/l) 1.5 0.8 3 Biomass (g/l) 0.6 1 2 Ethanol 0.4 0.5 1 Glucose 0.2 -10 10 20 30 XYN2 mRNA ACT1 mRNA -5 -2 1 3 6 18 12 Time (h) Expression of XYN2 under control of ADH2 promotor: Effect of pulse addition of glucose ( 5 g l-1) to an aerobic C-limited cont. culture of S. cerevisiae Y294

XYN2 mRNA ACT1 mRNA Time (h)
450 16 400 14 350 12 Intracellular xylanase activity (nkat/ml) 300 Specific rate of intracellular xylanase production, kat/(mg.h) 10 Extracellular xylanase activity (nkat/ml) 250 Extracellular xylanase production, nkat/(mg.h) Specific rate of 8 200 6 150 100 4 50 2 -10 -5 5 10 15 20 25 6 35 5 Biomass 30 25 4 Biomass (g/l) 20 Ethanol (g/l) 3 15 2 Ethanol 10 1 5 -10 -5 5 10 15 20 25 XYN2 mRNA ACT1 mRNA -5 -2 1 3 5 20 10 8 Time (h) Expression of XYN2 under control of ADH2 promotor: Effect of pulse addition of ethanol (30 g l-1) to an aerobic C-limited cont. culture of S. cerevisiae CEN.PK110-6C

Growth of S. cerevisiae ADH1 adh2 adh3 adh4 adh5 quadruple deletion mutant on glucose
Biomass Biomass Glucose Glucose Glycerol Acetate Glycerol Acetate Ethanol Ethanol ADH1 quadruple deletion mutant Strain W303-1A (wild type)

Growth of S. cerevisiae ADH2 adh1 adh3 adh4 adh5 quadruple deletion mutant on glucose
Biomass Glycerol Ethanol Biomass Acetaldehyde Glucose Glycerol Acetic acid Glucose Ethanol Parental strain ADH2 quadruple deletion mutant

Growth of S. cerevisiae ADH1 adh2 adh3 adh4 adh5 & ADH2 adh1 adh3 adh4 adh5 in batch culture on ethanol Q1 & Q2 Ethanol ADH2 Biomass ADH1 Biomass Biomass ADH2 Ethanol ADH1 Ethanol Parental strain ADH1 & ADH2 quadruple deletion mutants

Growth parameters of S. cerevisiae deletion mutants in batch culture on glucose
Wild type Only ADH1 ADH2 ADH3 ADH4 ADH5 max, h-1 0.44 0.45 0.21 0.19 0.18 Yx/s glucose 0.195 0.14 0.06 0.07 0.05 Yp/s, ethanol 0.38 0.37 0.13 0.11 Yp/s, glycerol 0.03 0.33 0.35 0.36 0.34 Acetaldehyde, g l-1 1.23 0.92 ?? µmax Maximum specific growth rate Yx/s Biomass yield coefficient (g dry biomass / g glucose utilised) Yp/s Product yield coefficient (g product / g glucose utilised)

Growth of S. cerevisiae Q1 and Q2 quadruple deletion mutants in batch culture
Carbon source Glucose Ethanol Parameter Parental strain Only ADH1 ADH2 max, h-1 0.44 0.45 0.21 0.20 0.195 Yx/s 0.14 0.07 0.50 Yp/s, ethanol 0.38 0.37 0.13 Yp/s, glycerol 0.03 0.33 Qpmax, g l-1 h-1 0.86 0.84 0.05 Glycerol, g l-1 0.48 0.32 3.1 Acetic acid, g l-1 0.22 0.17 Acetaldehyde, g l-1 1.23

Study the possible role of each enzyme in ethanol metabolism
Deletion constructs were constructed with standard molecular and classical techniques W303-1A adh1 to adh5 M Lane 1 padh1::LEU2 Lane 2 padh2::URA3 Lane 3 padh3::TRP1 Lane 4 padh4::HIS3 Lane 5 padh5::LEU2

Single deletion mutants produced in S
Single deletion mutants produced in S. cerevisiae W303-1A via one step transformation padh1::LEU2 padh2::URA3 padh3::TRP1 padh4::HIS3 padh5::LEU2 X X Recombination in yeast in vivo

Deletion mutant screening with PCR
2000 1500 1200 1031 W303-1A ADH 2000 1500 1200 1031 Quadruple deletion strain Q1 ADH 2U 3T 4H 5L

ethanol acetaldehyde acetate Acetyl-CoA acetyl-CoA pyruvate
lipids pyruvate oxaloacetate mitochondrion Pdh amino acids Pyc Pdc glucose glycolysis cytosol Adh Ald Acs TCA cycle PDH, pyruvate dehydrogenase complex; PYC, pyruvate carboxylase; PDC, pyruvate decarboxylase; ALD,acetaldehyde dehydrogenase; ACS, acetyl-coenzyme A synthetase; ADH, alcohol dehydrogenase Not certain exactly where the rate limiting reaction(s) might reside: Limited capacity of mitochondrial transport system for pyruvate Insufficient activity of the pyruvate dehydrogenase complex Limited capacity of TCA cycle enzymes Limited capacity of mitochondrial electron transport Flikweert et al. 1997, 1999

10 20 30 40 2 4 6 8 12 14 16 qO2 RQ qCO2 0.1 0.2 0.3 0.4 0.5 Biomass Ethanol Glucose Steady-state parameters for chemostat culture of S. cerevisiae CBS 8066 Biomass, Glucose & Ethanol, gl-1 Dilution rate (h-1) R.Q. qO2 (mmol.g .h-1), qCO2 (mmol.g .h-1)

A high homology between Adh1 and Adh2 (95%);
76-77% sequence identity with ADH5. Adh4 not homologous to the other yeast Adh.

The use of multiple deletion mutants for determining the physiological role of alcohol dehydrogenase isozymes in Saccharomyces cerevisiae James du Preez.

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The use of multiple deletion mutants for determining the physiological role of alcohol dehydrogenase isozymes in Saccharomyces cerevisiae James du Preez.

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