Regulatory aspects of bakers' yeast metabolism in aerobic fed-batch cultures

A highly instrumented computer-coupled bioreactor is used to investigate metabolic changes of Saccharomyces cerevisiae in aerobic fed-batch systems which are generally applied in bankers' yeast manufacture. The four types of metabolism (oxidation of glucose, aerobic fermentation, oxidation of glucose and ethanol, and oxidation of ethanol) appearing in such systems are characterized by four significant fermentation parameters: Respiratory quotient (RQ), glucose uptake rate (Q_g), ethanol turnover rate (Q_EtOH), and growth yield on glucose (Y_g). Below the critical glucose concentration glucose and ethanol are utilized simultaneously. The shift from aerobic fermentation to nondiauxic growth on glucose and ethanol is not only dependent on glucose concentration. but also on the precultivation on cells. The uptake of ethanol is controlled by the glucose supply except in the case when ethanol is limiting; the oxygen uptake rate (Q_o2), however, is unaffected by the ratio of Q_g and Q_EtOH. Critical glucose concentration is not a constant value for a particular strain, but varies corresponding to the nutritional state of the cells.     

Regulatory mechanisms of cellular respiration; the role of cell membranes; uranium inhibition of cellular respiration

Uranium as UO(2)(NO(3))(2) combines reversibly with proteins. The degree of dissociation of this combination depends, among other factors, on the H(+) concentration. At pH 7.3 the U-albumin complex was easily dissociated on addition of citrate, while at pH 3.8 it was not. Uranium inhibited reversibly a number of enzyme systems. Uranium enzyme inhibitions could be reversed on addition of certain hydroxypolycarboxylic acids (citric acid, alpha-hydroxyaspartic acid, malic acid); in no case, however, did phosphate have any effect. In cell-free yeast juice, the fermentation of glucose-hexosediphosphate was inhibited by UO(2)(NO(3))(2). Slight reactivation occurred on addition of phosphate. In living yeast cells, the fermentation and oxidation of glucose was inhibited by small amounts of UO(2)(NO(3))(2) (7,7 micrograms per mg. dry weight), while the oxidation of acetic acid, ethyl alcohol, malic and citric acids, was not affected at all. U inhibition in living yeast cells at pH 7.3 was completely released on addition of small amounts of phosphate, adenosinetriphosphate, and citrate, while at pH 3.8 U inhibition was not released by phosphate and citrate. At saturation, one yeast cell contained 7.06 x 10(6) molecules of uranium. Lactic dehydrogenase was not inhibited by U while the oxidation of lactic acid by gonococci was inhibited. Addition of phosphate released this inhibition. The U inhibition of liver succinoxidase was unaffected by phosphate, while the U inhibition of the oxidation of succinate by E. coli was released by phosphate. It has been concluded from these experiments that U inhibition of cell metabolism is due to combination of the metal with the protein portion of the cell membrane. Uranium is presented as an example of surface inhibition.     

Fatty acid cellular metabolism and lactone production by the yeast Pichia guilliermondii

Methyl ricinoleate conversion into γ-decalactone by fungi is already widely used by the aromatic industry. It offers an interesting alternative to chemical synthesis by permitting acquisition of a natural label. Peroxisomal β-oxidation has been described as the probable transformation mechanism. This paper provides information about this metabolism and shows the importance of the step catalysed by carnitine octanoyltransferase. After culture of the yeast Pichia guilliermondii on a medium containing methyl ricinoleate as sole carbon source, we confirmed that mitochondrial β-oxidation could not be responsible for the biotransformation. We also observed the effect of chlorpromazine, an inhibitor of carnitine octanoyltransferase, on peroxisomal β-oxidation and therefore on lactone production, and on lipid accumulation by the yeasts. The presence of chlorpromazine caused a reduction in aromatic specific production yield. This reduction was inversely proportional to the amount of chlorpromazine present in the medium. A considerable accumulation of methyl ricinoleate derivatives was also observed. We therefore concluded that the metabolism responsible for the bioconversion was peroxisomal β-oxidation. The effects of chlorpromazine suggested that the entry of fatty acids into the peroxisomes took place in a carnitine-dependent manner. This step might be a limiting step in the metabolism. Received: 26 June 1995/Received revision: 16 November 1995/Accepted: 4 December 1995     

Regulatory mechanisms of cellular respiration; the role of soluble sulfhydryl groups as shown by the effect of sulfhydryl reagents on the respiration of sea urchin sperm

Oxidizing agents of sulfhydryl groups such as iodosobenzoate, alkylating agents such as iodoacetamide, and mercaptide-forming agents such as cadmium chloride, mercuric chloride, p-chloromercuribenzoate, sodium arsenite, and p-carboxyphenylarsine oxide, added in small concentrations to a suspension of sea urchin sperm produced an increase in respiration. When the concentration was increased there was an inhibition. These effects are explained by postulating the presence in the cells of two kinds of sulfhydryl groups: soluble sulfhydryl groups, which regulate cellular respiration, and fixed sulfhydryl groups, present in the protein moiety of enzymes. Small concentrations of sulfhydryl reagents combine only with the first, thus producing an increase in respiration; when the concentration is increased, the fixed sulfhydryl groups are also attacked and inhibition of respiration is the consequence. Other inhibitors of cell respiration, such as cyanide and urethanes, which do not combine with -SH groups, did not stimulate respiration in small concentration.     

Enzyme activities of D-glucose metabolism in the fission yeast Schizosaccharomyces pombe.

The activities of key enzymes that are members of D-glucose metabolic pathways in Schizosaccharomyces pombe undergoing respirative, respirofermentative, and fermentative metabolisms are monitored. The steady-state activities of glycolytic enzymes, except phosphofructokinase, decrease with a reduced efficiency in D-glucose utilization by yeast continuous culture. On the other hand, the enzymic activities of pentose monophosphate pathway reach the maximum when the cell mass production of the cultures is optimum. Enzymes of tricarboxylate cycle exhibit the maximum activities at approximately the washout rate. The steady-state activity of pyruvate dehydrogenase complex increases rapidly when D-glucose is efficiently utilized. By comparison, the activity of pyruvate decarboxylase begins to increase only when ethanol production occurs. Depletion of dissolved oxygen suppresses the activity of pyruvate dehydrogenase complex but facilitates that of pyruvate decarboxylase. Acetate greatly enhances the acetyl CoA synthetase activity. Similarly, ethanol stimulates alcohol dehydrogenase and aldehyde dehydrogenase activities. Evidence for the existence of alcohol dehydrogenase isozymes in the fission yeast is presented.     

The influence of yeast metabolism on dispersion in a fluidised yeast bed

Summary The effect of yeast metabolism on the dispersion characteristics of a fluidised bed fermentor containing flocs of the yeast Saccharomyces carlsbergensis was investigated. Dispersion in the metabolizing fluidised yeast floc system was compared with the dispersion in an inert yeast floc system and in a glass bead system. Breakdown in plug-flow was found to occur in the metabolically active yeast bed when the flow rate was increased over a relatively narrow operating range (up to a dilution rate of 0.08 h^-1). The superficial liquid velocity at which perfect mixing was approximated was some 18 times greater in the inert yeast floc system than in the metabolizing yeast floc system.Abbreviations C Tracer concentration - C ₀ Concentration of tracer at time t=0 - V Mixing chamber volume - v Volumetric flow rate - l Time - t Mean residence time - N Number of tanks in series - D/l Dispersion number - ² variance     

Cytochrome c1 of bakers' yeast. II. Synthesis on cytoplasmic robosomes and influence of oxygen and heme on accumulation of the apoprotein.

In the preceding paper (Ross, E., and Schatz, G. (1976) J. Biol. Chem. 251, 1991-1996) yeast cytochrome c1 was characterized as a 31,000 dalton polypeptide with a covalently bound heme group. In order to determine the site of translation of this heme-carrying polypeptide, yeast cells were labeled with [H]leu(be under the following conditions: (a) in the absence of inhibitors, (b) in the presence of acriflavin (an inhibitor of mitochondrial translation), or (c) in the presence of cycloheximide (an inhibitor of cytoplasmic translation). The incorporation of radioactivity into the hemeprotein was measured by immunoprecipitating it from mitochondrial extracts and analyzing it by dodecyl sulfate-polyacrylamide gel electrophoresis. Label was incorporated into the cytochrome c1 apoprotein only in the presence of acriflavin or in the absence of inhibitor, but not in the presence of cycloheximide. Cytochrome c1 is thus a cytoplasmic translation product. This conclusion was further supported by the demonstration that a cytolasmic petite mutant lacking mitochondrial protein synthesis still contained holocytochrome c1 that was indistinguishable from cytochrome c1 of wild type yeast with respect to molecular weight, absorption spectru, the presence of a covalently bound heme group, and antigenic properties. Cytochrome c1 in the mitochondria of the cytoplasmic petite mutant is firmly bound to the membrane, and its concentration approaches that typical of wild type mitochondria. However, its lability to proteolysis appeared to be increased. A mitochondrial translation product may thus be necessary for the correct conformation or orientation of cytochrome c1 in the mitochondrial inner membrane. Accumulation of cytochrome c1 protein in mitochondria is dependent on the abailability of heme. This was shown with a delta-aminolevulinic acid synthetase-deficient yeast mutant which lacks heme and any light-absorbing peaks attributable to cytochromes. Mitochondria from mutant cells grown without added delta-aminolevulinic acid contained at least 20 times less protein immunoprecipitable by cytochrome c1-antisera than mitochondria from cells grown in the presence of the heme precursor. Similarly, the respiration-deficient promitochondria of anaerobically grown wild type cells are almost completely devoid of material cross-reacting with cytochrome c1-antisera. A 105,000 X g supernatant of aerobically grown wild type cells contains a 29,000 dalton polypeptide that is precipitated by cytochrome c1-antiserum but not by nonimmune serum. This polypeptide is also present in high speed supernatants from the heme-deficient mutant or from anaerobically gorwn wild type cells. The possible identity of this polypeptide with soluble apocytochrome c1 is being investigated.