1. Phenotype microarray analysis of the metabolic profiles of multiple ADH deletion mutants of S. cerevisiae
Laurinda Steyn1, James du Preez1, Jacobus Albertyn1 and Olga de Smidt2
1Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, PO Box 339, Bloemfontein 9300, South Africa.
2School of Agriculture and Environmental Sciences, Central University of Technology, Bloemfontein 9300, South Africa
Introduction
Results
Five alcohol dehydrogenase genes (ADH) in Saccharomyces cerevisiae are associated with ethanol metabolism in the glycolytic pathway and are also involved in the production of fusel alcohols via the Ehrlich pathway, with the reduction of NADH to NAD+ (Fig 1). (1, 2, 3, 4, 5, 6)
The objective of this study was to elucidate the metabolic effects of multiple ADH gene deletion mutants and a null mutant, using a high-throughput analytical approach. The metabolic profiles were determined using an OmniLog® Phenotype MicroArray™ system, which is based on redox chemistry and employs cell respiration as a universal reporter. The assay facilitates the rapid and precise quantification of phenotypes.
A complement of alcohol dehydrogenase (Adh) isozymes is required for normal metabolic function in S. cerevisiae. Mutants lacking some of the ADH genes are more sensitive to osmotic stress.
Phenotypic analysis provided a deeper understanding of the possible functional substitution of these five Adh isozymes by comparing the lost or gained ability of the mutant strains to utilise substrates compared to the reference strain (Fig. 2). Mutants with Adh isozymes 1 or 2 as the only functional Adh exhibited carbon substrate utilisation profiles similar to the reference strain, but differed in respect of nitrogen and phosphorus substrate utilisation. The substrate utilisation profiles of mutants with Adh isozymes 3, 4 or 5 as the only functional Adh differed significantly from the reference strain, but were similar to one another with only slight differences in the utilisation of a few substrates; all were sensitive to osmotic stress. The amino acid substrate utilisation profiles of the mutant strains, in contrast to the reference strain, indicated little or no utilisation of these substrates.
C- sources
N-, P- and S-sources
(incl. amino acid as N-source)
Peptide-sources
Osmotic, toxicity and pH sensitivity
W303-1A Q1 Q2 Q3 Q4 Q5 Q0
287
137 92 90
106 84 91
115
118
129
143 97
102
112
108
242 27 3 4 5 10
237
114 63 68 79 65 67 46 1
209
100 56 53 61 45 51 18
200 43 41 49 39 36
101 6
180 37 31 26 57 8 21 2 52
179 17
109
128 93 54 44 30 38 9
170
234 13 7 25 55 40
247 12 11 87 66 14
144
182 48 32 19 35 15 50 23 22 20 59 47 28 16 60
233 42
152 34
156 77
221
153 85 33
181
238
164 94
216
168
235
136
166
132 95
218
171
208
219
103
191
217
147
105
212 58
183 29
190
184
145 76
150
177
155
139
158
175
231
213
126 62 64 83
173
193
176 96
110
169
149
163
228
113 89 78
116 82
199 99 75 86
111
339
224
104 72
336
222 74 88 81 73
327 70
325
227
343
307
174
332
293
260
178
317
276
134
172
159
151
308
240
125
141
344
239
123
121
319
194
321
310
201
244
274
124
271
225
278 69 24
119
304 98
268
269
165
160
223
245
229
236
140
215
142
210 71 80
352
291
250
345
256
246
252
295
196
243
350
299
107
253
272
206
133
122
363
226
146
266
357
135
162
214
360
248
197
161
251
358
211
204
117
148
127
186
205
220
192
230
249
254
261
267
265
273
188
185
187
207
131
202
281
279
286
138
262
198
284
203
257
167
157
130
264
255
154
120
259
275
300
283
285
292
232
241
DHAP
NAD+
NADH
Glucose
Glyceraldehyde-3-P
1,3-P-Glycerate
Glycolysis
Ethanol
Pentose Phosphate Pathway
Pyruvate
NAD(P)H
NAD(P)+
Amino acid
ADH
a-keto acid
Fusel aldehyde
Fusel acid
Fusel alcohol
CO2
NADH, H+
Ehrlich Pathway
TCA cycle
a-ketoglutarate
glutamate
Acetate
Ethanol Metabolism
TCA Cycle
Acetaldehyde
Amino acids
Fig. 1 The involvement of alcohol dehydrogenase (ADH) genes in the glycolytic and Ehrlich pathways with reduction of NADH to NAD+ (2, 4) .
Materials & Methods
S. cerevisiae strains used in this study, where the number after Q indicates the intact ADH gene in the genetically engineered quadruple deletion mutants (7). Q0 denotes the null mutant.
Cultivations were done in microtitre plates; each well pre-filled with a different desiccated substrate. Automated microplate incubation and capture of phenotypic data by OmniLog® instrument.
Yeast strain
Genotype
W303-1A (auxotrophic host)
W303-1A, MATa, his3, leu2, trp1, ura3 Q1
W303-1A, MATa, ADH1, adh2∆::URA3, adh3∆::TRP1, adh4∆::HIS3, adh5∆::LEU2 Q2
W303-1A, MATa, adh1∆::URA3, ADH2, adh3∆::TRP1, adh4∆::HIS3, adh5∆:: LEU2 Q3
W303-1A, MATa, adh1∆::TRP1, adh2∆::URA3, ADH3, adh4∆::HIS3, adh5∆::LEU2 Q4
W303-1A, MATa, adh1∆::HIS3, adh2∆::URA3, adh3∆::TRP1, ADH4,adh5∆:: LEU2 Q5
W303-1A, MATa, adh1∆::LEU2, adh2∆::URA3 adh3∆::TRP1, adh4∆::HIS3, ADH5 Q0
W303-1A, MATa, adh1∆::HIS3, adh2∆::URA3, adh3∆::TRP1, adh4∆::KanMXb, adh5∆:: LEU2
Fig. 2 Heat profiles of the maximum metabolic profiles of S. cerevisiae strains on the different substrates supplied in the microtitre plates of the OmniLog® Phenotype MicroArray™ system, where red indicates the highest activity and green no or little activity.
On closer inspection of the carbon substrate profiles, similar profiles were observed for ribose and pyruvic acid (Table 1). These results suggest that when no additional NADH was produced in the glycolytic pathway, Adh isozymes were not required for restoring the redox balance. Restoring the redox balance via ethanol metabolism was, however, required during metabolism of glucose or galactose. It was curious that, during glucose metabolism by mutant Q2, the metabolic activity was similar to that of the reference strain. This observation suggested that this ADH2 gene was important during ethanol metabolism: not only during the metabolism of ethanol as substrate, but also during the metabolism of glucose to ethanol.
Table 1 Maximum metabolic values of some of the carbon substrates of the S. cerevisiae strains.
Carbon substrate
Yeast strain
W303-1A Q1 Q2 Q3 Q4 Q5 Q0
d-Galactose 57 8 21 3 6 5
d-Ribose
155
152
190
219
139
145
158
a-d-Glucose
101 13 96 2 7
Pyruvic acid
181 84 81 71 62 61 43
References
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Conclusions
A complement of Adh isozymes is required for normal metabolic function in S. cerevisiae .
Mutants without all Adh isozymes are more sensitive to osmotic stress.
Adh1p alone is not sufficient for normal metabolic function on glucose as substrate.
Only Adh5p plus Adh6p activities in the Ehrlich pathway for conversion of aldehydes to alcohols are insufficient for amino acid utilisation.
The inability of mutant strains to utilise amino acids could possibly be due to the accumulation of fusel aldehydes and their resulting toxic effect.
Acknowledgements
The National Research Foundation, South Africa for financial support
Dr. George Charimba for able technical assistance with the OmniLog® Phenotype MicroArray™ system