Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae.

This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates. Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol. Therefore we constructed mutants in two parental strains (YPH499 and W303-1a). Some differences appeared in the growth characteristics of mutants obtained from these two parental strains. For these experiments we used ethanol, pyruvate or lactate as substrates. Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway. The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain. The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent. To understand these differences, we measured the enzyme content of these different strains. We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase. Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation. Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient. However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation.     

Effects of magnesium, manganese and adenosine triphosphate ions on pyruvate carboxylase from baker's yeast

1. Pyruvate carboxylase from baker's yeast acts with either MgATP(2-) or MnATP(2-) as substrate. The optimum pH for the enzyme reaction is 8.0 with MgATP(2-) and 7.0 with MnATP(2-). 2. When the reaction velocity is plotted against MgATP(2-) (or MnATP(2-)) concentration slightly sigmoid curves are obtained, either in the presence or in the absence of acetyl-CoA (an allosteric activator). In the presence of excess of free Mg(2+) (or Mn(2+)) the curves turn into hyperbolae, whereas in the presence of excess of free ATP(4-) the apparent sigmoidicity of the curves increases. 3. The sigmoidicity of the plots of v against MgATP(2-) (or MnATP(2-)) concentration can be explained by the inhibitory effect of free ATP(4-), the concentration of which, in the experimental conditions employed, is significant and varies according to the total concentration of the ATP-magnesium chloride (or ATP-manganese chloride) mixture. Free ATP(4-) behaves as a negative modifier of yeast pyruvate carboxylase. 4. The effect of high concentrations of Mg(2+) (or Mn(2+)) on the kinetics of yeast pyruvate carboxylase can be explained as a deinhibition with respect to ATP(4-), instead of a direct enzyme activation. 5. At pH6.5 manganese chloride is more effective than magnesium chloride as enzyme activator even in the presence of a great excess (16-fold) of the latter. This is consistent with a significant contribution of the MnATP(2-) complex to the activity of yeast pyruvate carboxylase, in medium conditions resembling those existing inside the yeast cell (pH6.25-6.75; 12mm-magnesium chloride and 0.75mm-manganese chloride). 6. The physiological significance of the enzyme inhibition by free ATP(4-) is doubtful since the Mg(2+) and Mn(2+) concentrations reported to exist inside the yeast cell are sufficient to decrease ATP(4-) concentrations to ineffective values.     

Indications of pH-induced conformational changes in phosphofructokinase from baker's yeast.

The partition of phosphorfructokinase (EC 2.7.1.11) from baker's yeast between the liquid phases of an aqueous biphasic systeem changes drastically in the pH interval 7-8, in contrast to other proteins. This abnormal behaviour is correlated to changes in sedimentation coefficient and binding capacity of the enzyme in this pH region. Since the molecular weight of phosphofructokinase does not change, these findings must reflect conformational changes in the enzyme molecule.     

Effect of vitamin concentration on the synthesis of lactate, ethanol, pyruvate, and ethyl acetate in cells of the yeast Dipodascus magnusii

In the yeast Dipodascus magnusii, which is auxotrophic for thiamine and biotin, during cultivation on glucose with excessive thiamine concentration, pyruvate metabolism was shown to result in the synthesis of fermentation products, namely, ethanol and, to a lesser extent, lactate. Substantial synthesis of ethyl acetate was also observed under these conditions. Introduction of nicotinic acid (NA) into the medium resulted in time separation of ethanol and lactate production. It was shown that cultivation of the yeast under biotin deficiency resulted in nearly complete suppression of aerobic production of ethanol and cessation of ethyl acetate synthesis, whereas lactate synthesis was activated as early as in the first hours of cultivation. Upon introduction of NA under these conditions, lactate concentration sharply increased. These results show that the combination of thiamine and biotin with other vitamins can stimulate utilization of the pyruvate pool in yeasts towards formation of considerable amounts of lactate, which is typical only of cells of higher eukaryotes and bacteria.     

Effect of vitamin concentration on the synthesis of lactate, ethanol, pyruvate, and ethyl acetate in cells of the yeast Dipodascus magnusii

In the yeast Dipodascus magnusii, which is auxotrophic for thiamine and biotin, during cultivation on glucose with excessive thiamine concentration, pyruvate metabolism was shown to result in the synthesis of fermentation products, namely, ethanol and, to a lesser extent, lactate. Substantial synthesis of ethyl acetate was also observed under these conditions. Introduction of nicotinic acid (NA) into the medium resulted in time separation of ethanol and lactate production. It was shown that cultivation of the yeast under biotin deficiency resulted in nearly complete suppression of aerobic production of ethanol and cessation of ethyl acetate synthesis, whereas lactate synthesis was activated as early as in the first hours of cultivation. Upon introduction of NA under these conditions, lactate concentration sharply increased. These results show that the combination of thiamine and biotin with other vitamins can stimulate utilization of the pyruvate pool in yeasts towards formation of considerable amounts of lactate, which is typical only of cells of higher eukaryotes and bacteria.     

Utilization and metabolic effects of acetaldehyde and ethanol in the perfused rat liver

1. The formation of the two 16-unsaturated alcohols 5alpha-androst-16-en-3alpha-ol and 5alpha-androst-16-en-3beta-ol from [5alpha-(3)H]5alpha-androst-16-en-3-one has been demonstrated in boar testis homogenates. 2. The optimum yield (23%) of the 3alpha-alcohol was obtained in the presence of NADPH, whereas that for the 3beta-alcohol (74%) was obtained when NADH was the added cofactor. 3. The two alcohols were not interconvertible. 4. Prolonged storage of boar testis tissue at -20 degrees C abolished the ability to form all androst-16-enes except androsta-4,16-dien-3-one from [4-(14)C]progesterone. 5. The production of 5alpha-androst-16-en-3-one and the two alcohols from [7alpha-(3)H]androsta-4,16-dien-3-one only occurred when fresh tissue was used, whereas reduction of [5alpha-(3)H]5alpha-androst-16-en-3-one was unaffected by storage of testis at -20 degrees C. 6. NADPH was the preferred cofactor for the reduction of androsta-4,16-dien-3-one. 7. The previously established conversion of androsta-5,16-dien-3beta-ol into androsta-4,16-dien-3-one was shown to be reversible, NADH and NADPH being equally effective cofactors. 8. Pathways of biosynthesis of 5alpha-androst-16-en-3alpha- and 3beta-ols, with the C(19) 3-oxo steroids as intermediates, are presented.     

Purification and properties of phosphorylase from baker's yeast

A rapid, reliable method for purification of phosphorylase, yielding 200-400 mg pure phosphorylase from 8 kg of pressed baker's yeast, is described. The enzyme is free of phosphorylase kinase activity but contains traces of phosphorylase phosphatase activity. Phosphorylase constitutes 0.5-0.8% of soluble protein in various strains of yeast assayed immunochemically. The subunit molecular weight (Mr) of yeast phosphorylase is around 100,000. The enzyme is composed of two subunits in various ratios, differing slightly in molecular weight and N-terminal sequence. Both are active. Only the enzyme species containing the larger subunit can form tetramers and higher oligomers. The activated enzyme is dimeric. Correlated with specific activity (1 to 110 U/mg), phosphorylase contained between less than 0.1 to 0.74 covalently bound phosphate per subunit. Inactive forms of phosphorylase could be activated by phosphorylase kinase and [gamma-32P]ATP with concomitant phosphorylation of a single threonine residue in the aminoterminal region of the large subunit. The small subunit was not labeled. The incorporated phosphate could be removed by yeast phosphorylase phosphatase, resulting in loss of activity of phosphorylase, which could be restored by ATP and phosphorylase kinase.     

Replicative inactivation and metabolic inhibition in yeast ethanol fermentations

Summary The effects of ethanol on the growth rates of twoSaccharomyces yeast strains were measured during normal batch fermentative growth and compared with those measured by initial rate studies. In the light of previous work, which has highlighted the loss of cell replicative ability caused by ethanol, the results imply that the observed reduction in growth rate reflects a mixture of true inhibition and replicative inactivation.     

Kinetics of ethanol production by baker's yeast in an integrated process of fermentation and microfiltration

The productivity of a fermentation is proportional to the biomass concentration. The productivity can therefore be increased by retention of the cells in the fermentor. In this study microfiltration was used for cell retention in a fermentation of glucose to ethanol by baker's yeast. Compared to a system without cell retention the productivity could be increased 12-fold to 55 kg/m³ h at a biomass concentration of 135 kg/m³. Maximal ethanol concentrations of 76 kg/m³ were obtained at conditions of growth. At zero growth conditions in the integrated system the ethanol concentration could be increased to about 115 kg/m³, and could be produced for at least 10 hours. The fermentation results in the integrated system could be described reasonably well with a mathematical model based on a different linear inhibition kinetics for growth and substrate consumption.     

Application of fuzzy reasoning to control of glucose and ethanol concentrations in baker's yeast culture

Fuzzy reasoning was applied to control both ethanol and glucose concentrations in fed-batch cultures of baker's yeast. This fuzzy controller consisted of three membership functions (concentrations of dissolved oxygen (DO), ethanol and glucose) and 18 production rules. Fuzzy inference was carried out by IF {A is a and B is b,...#x007D;, THEN {C is c} from the on-line measured concentrations of DO, ethanol and glucose. When medium concentrations of ethanol and glucose in fed-batch culture of baker's yeast were set at 2 g/l and 0.2 g/l, both ethanol and glucose concentrations were controlled at 2.67±0.35 g/l and 0.27±0.25 g/l, respectively, ethanol production was reduced from 26 g/l to 34 g/l, cell yield increased from 0.38 to 0.53 g dry cell/g consumed glucose and ethanol yield decreased from 0.50 to 0.14 g ethanol/g consumed glucose, respectively, as compared with those of the glucose only control at 0.2 g/l.     

Shift in product formation from acetate to ethanol using metabolic inhibitors inFusarium oxysporum

Summary Fusarium oxysporum 841 produces a mixture of ethanol and acetic acid from glucose, xylose or Avicel (microcrystalline cellulose) substrates. Some metabolic inhibitors viz. sodium azide, dinitrophenol and polyethylene glycol were used for shifting product formation from acetic acid to ethanol. Using these inhibitors 1.5- to 2- fold increase in ethanol production was achieved with significant repression (by 80 to 90%) of acetic acid. Almost theoretical yields of ethanol were obtained.     

Artifactual production and recovery of acetaldehyde from ethanol in urine

Ethanol (1575 mg/L) incubated with fresh urine from healthy, ethanol-free subjects yields acetaldehyde. The concentration of acetaldehyde depends upon temperature, time of incubation, and pH. In samples made hypertonic with sodium chloride (200 mg/mL) and in samples filtered through 0.45-micron membranes, acetaldehyde production was not decreased. L-Ascorbic acid (0.1 mg/mL) added to normal pooled urine caused a threefold increase in acetaldehyde production but thiourea (7.6 mg/mL) stopped it. This suggests that the oxidation of ethanol to acetaldehyde is catalyzed by the semidehydroascorbate peroxy radical of ascorbic acid. Recovery of acetaldehyde added to urine was less than 100% over the pH range 1.5 to 10. Relative to blood, artifactual production of acetaldehyde from ethanol in urine is more easily controlled and is up to an order of magnitude less but corrections for the variables above are still required.