Physiological characterisation of a pyruvate-carboxylase-negative Saccharomyces cerevisiae mutant in batch and chemostat cultures

A prototrophic pyruvate-carboxylase-negative (Pyc-) mutant was constructed by deleting the PYC1 and PYC2 genes in a CEN.PK strain of Saccharomyces cerevisiae. Its maximum specific growth rate on ethanol was identical to that of the isogenic wild type but it was unable to grow in batch cultures in glucose-ammonia media. Consistent with earlier reports, growth on glucose could be restored by supplying aspartate as a sole nitrogen source. Ethanol could not replace aspartate as a source of oxaloacetate in batch cultures. To investigate whether alleviation of glucose repression allowed expression of alternative pathways for oxaloacetate synthesis, the Pyc- strain and an isogenic wild-type strain were grown in aerobic carbon-limited chemostat cultures at a dilution rate of 0.10 h-1 on mixtures of glucose and ethanol. In such mixed-substrate chemostat cultures of the Pyc- strain, steady-state growth could only be obtained when ethanol contributed 30% or more of the substrate carbon in the feed. Attempts to further decrease the ethanol content of the feed invariably resulted in washout. In Pyc- as well as in wild-type cultures, levels of isocitrate lyase, malate synthase and phospho-enol-pyruvate carboxykinase in cell extracts decreased with a decreasing ethanol content in the feed. Nevertheless, at the lowest ethanol fraction that supported growth of the Pyc- mutant, activities of the glyoxylate cycle enzymes in cell extracts were still sufficient to meet the requirement for C4-compounds in biomass synthesis. This suggests that factors other than glucose repression of alternative routes for oxaloacetate synthesis prevent growth of Pyc-mutants on glucose.     

Growth requirements of pyruvate-decarboxylase-negative Saccharomyces cerevisiae

Pyruvate-decarboxylase (Pdc)-negative Saccharomyces cerevisiae has been reported to grow in batch cultures on glucose-containing complex media, but not on defined glucose-containing media. By a combination of batch and chemostat experiments it is demonstrated that even in complex media, Pdc- S. cerevisiae does not exhibit prolonged growth on glucose. Pdc- strains do grow in carbon-limited cultures on defined media containing glucose-acetate mixtures. The acetate requirement for glucose-limited growth, estimated experimentally by continuously decreasing the acetate feed to chemostat cultures, matched the theoretical acetyl-CoA requirement for lipid and lysine synthesis, consistent with the proposed role of pyruvate decarboxylase in the synthesis of cytosolic acetyl-CoA.     

In vivo kinetics of primary metabolism in Saccharomyces cerevisiae studied through prolonged chemostat cultivation

In this study, prolonged chemostat cultivation is applied to investigate in vivo enzyme kinetics of Saccharomyces cerevisiae. S. cerevisiae was grown in carbon-limited aerobic chemostats for 70-95 generations, during which multiple steady states were observed, characterized by constant intracellular fluxes but significant changes in intracellular metabolite concentrations and enzyme capacities. We provide evidence for two relevant kinetic mechanisms for sustaining constant fluxes: in vivo near-equilibrium of reversible reactions and tight regulation of irreversible reactions by coordinated changes of metabolic effectors. Using linear-logarithmic kinetics, we illustrate that these multiple steady-state measurements provide linear constraints between elasticity parameters instead of their absolute values. Upon perturbation by a glucose pulse, glucose uptake and ethanol excretion in prolonged cultures were remarkably lower, compared to a reference culture perturbed at 10 generations. Metabolome measurements during the transient indicate that the differences might be due to a reduced ATP regeneration capacity in prolonged cultures.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Effects of growth rate and influent substrate concentration on effluent quality from chemostats containing bacteria in pure and mixed culture

Studies were performed using pure cultures of A. acrogenes and E. coli and a heterogeneous microbial population growing in carbon-limited chemostats with glucose as the sole carbon and energy source. A two-level factorial experimental design was employed to test the hypothesis that the concentration of growth-limiting substrate in a chemostat is controlled by the growth rate alone and is independent of the concentration of substrate entering the reactor. The pure culture experiments showed that the conclusions depend upon the measurement employed for growth-limiting substrate. When the concentration of glucose was measured directly, the hypothesis was found to be true within the limits of the study (500–1500 mg/liter). However, if the chemical oxygen demand (COD) test was used as the measure of growth-limiting substrate the hypothesis was found to be false. When heterogeneous cultures were employed the hypothesis was false regardless of the technique used to measure the concentration of growth-limiting substrate. Nevertheless, it was possible to generate regression equations which described the interactions among influent COD, growth rate, and effluent COD with a high level of correlation.     

Selection of glucose-assimilating variants ofAcinetobacter calcoaceticus LMD 79.41 in chemostat culture

Glucose metabolism has been studied in two strains ofAcinetobacter calcoaceticus. Strain LMD 82.3, was able to grow on glucose and possessed glucose dehydrogenase (EC 1.1.99.17). Glucose oxidation by whole cells was stimulated by PQQ, the prosthetic group of glucose dehydrogenase. PQQ not only increased the rate of glucose oxidation and gluconic acid production but also shortened the lag phase for growth on glucose. Strain LMD 79.41 also possessed glucose dehydrogenase but was unable to grow on glucose. Batch cultures and carbon-limited chemostat cultures growing on acetate in the presence of glucose oxidized the sugar to gluconic acid, which was not further metabolized. However, after prolonged cultivation on mixtures of acetate and glucose, carbon-limited chemostat cultures suddenly acquired the capacity to utilize gluconate. This phenomenon was accompanied by the appearance of gluconate kinase and a repression of isocitrate lyase synthesis. In contrast to the starter culture, cells from chemostats which had been fully adapted to gluconate utilization, were able to utilize glucose as a sole carbon and energy source in liquid and solid media.     

Membrane enzymes associated with the dissimilation of some citric acid cycle substrates and production of extracellular oxidation products in chemostat cultures of Pseudomonas fluorescens

Enzyme activities forming extracellular products from succinate, fumarate, and malate were examined using washed cell suspensions of Pseudomonas fluorescens from chemostat cultures. Membrane-associated enzyme activities (glucose, gluconate, and malate dehydrogenases), producing large accumulations of extracellular oxidation products in carbon-excess environments, have previously been found in P. fluorescens. Investigations carried out here have demonstrated the presence in this microorganism of a malic enzyme activity which produces extracellular pyruvate from malate in carbon-excess environments. Although the three membrane dehydrogenase enzymes decrease significantly in carbon-limited chemostat cultures, malic enzyme activity was found to increase fourfold under these conditions. The regulation of malate dehydrogenase and malic enzyme by malate or succinate was similar. Malate dehydrogenase increased and malic enzyme decreased in carbon-excess cultures. The opposite effect was observed in carbon-limited cultures. When pyruvate or glucose was used as the carbon source, malate dehydrogenase was regulated similarly by the available carbon concentration, but malic enzyme activity producing extracellular pyruvate was not detected. While large accumulations of extracellular oxalacetate and pyruvate were produced in malate-excess cultures, no extracellular oxidation products were detected in succinate-excess cultures. This may be explained by the lack of detectable activity for the conversion of added external succinate to extracellular fumarate and malate in cells from carbon-excess cultures. In cells from carbon-limited (malate or succinate) cultures, very active enzymes for the conversion of succinate to extracellular fumarate and malate were detected. Washed cell suspensions from these carbon-limited cultures rapidly oxidized added succinate to extracellular pyruvate through the sequential action of succinate dehydrogenase, fumarase, and malic enzyme. Succinate dehydrogenase and fumarase activities producing extracellular products were not detected in cells from chemostat cultures using pyruvate or glucose as the carbon source. Uptake activities for succinate, malate, and pyruvate also were found to increase in carbon-limited (malate or succinate) and decrease in carbon-excess cultures. The role of the membrane-associated enzymes forming different pathways for carbon dissimilation in both carbon-limited and carbon-excess environments is discussed.     

Growth dynamics of Agrobacterium tumefaciens in chemostat cultures limited by carbon source and mineral nutrients.

Agrobacterium tumefaciens was grown in a chemostat in a chemically-defined medium which hs alpha-methyl D-glucoside, magnesium, manganese, phosphate or urea as the growth-limiting nutrient. Steady-state biomass concentrations were dependent on the specific growth rate of the organism when alpha-methyl D-glucoside, manganese or phosphate were growth-limiting nutrients. During magnesium-limited growth, large undamped oscillations in biomass concentration occurred. In all chemostat cultures a variant organism was selected which had lost the ability to grow in the medium supplied, bur survived on products of carbon metabolism derived from the wild-type.     

Intracellular carbon fluxes in riboflavin-producing Bacillus subtilis during growth on two-carbon substrate mixtures

Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional (13)C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.     

Influence of carbon source on α-amylase production by Aspergillus oryzae

The influence of the carbon source on alpha-amylase production by Aspergillus oryzae was quantified in carbon-limited chemostat cultures. The following carbon sources were investigated: maltose, maltodextrin (different chain lengths), glucose, fructose, galactose, sucrose, glycerol, mannitol and acetate. A. oryzae did not grow on galactose as the sole carbon source, but galactose was co-metabolized together with glucose. Relative to that on low glucose concentration (below 10 mg/l), productivity was found to be higher during growth on maltose and maltodextrins, whereas it was lower during growth on sucrose, fructose, glycerol, mannitol and acetate. During growth on acetate there was no production of alpha-amylase, whereas addition of small amounts of glucose resulted in alpha-amylase production. A possible induction by alpha-methyl-D-glucoside during growth on glucose was also investigated, but this compound was not found to be a better inducer of a-amylase production than glucose. The results strongly indicate that besides acting as a repressor via the CreA protein, glucose acts as an inducer.     

Chemostat cultivation as a tool for studies on sugar transport in yeasts.

Chemostat cultivation enables investigations into the effects of individual environmental parameters on sugar transport in yeasts. Various means are available to manipulate the specific rate of sugar uptake (qs) in sugar-limited chemostat cultures. A straightforward way to manipulate qs is variation of the dilution rate, which, in substrate-limited chemostat cultures, is equal to the specific growth rate. Alternatively, qs can be varied independently of the growth rate by mixed-substrate cultivation or by variation of the biomass yield on sugar. The latter can be achieved, for example, by addition of nonmetabolizable weak acids to the growth medium or by variation of the oxygen supply. Such controlled manipulation of metabolic fluxes cannot be achieved in batch cultures, in which various parameters that are essential for the kinetics of sugar transport cannot be controlled. In sugar-limited chemostat cultures, yeasts adapt their sugar transport systems to cope with the low residual sugar concentrations, which are often in the micromolar range. Under the conditions, yeasts with high-affinity proton symport carriers have a competitive advantage over yeasts that transport sugars via facilitated-diffusion carriers. Chemostat cultivation offers unique possibilities to study the energetic consequences of sugar transport in growing cells. For example, anaerobic, sugar-limited chemostat cultivation has been used to quantify the energy requirement for maltose-proton symport in Saccharomyces cerevisiae. Controlled variation of growth conditions in chemostat cultures can be used to study the differential expression of genes involved in sugar transport and as such can make an important contribution to the ongoing studies on the molecular biology of sugar transport in yeasts.     

Studies of the Utilization of Coconut Water Waste for the Production of the Food Yeast Saccharomyces fragilis

The accepted food yeast Saccharomyces fragilis was grown in batch and chemostat culture on coconut water and on a simulated coconut-water medium containing glucose, fructose, sucrose and sorbitol, to provide kinetic data for a feasibility study of microbial protein production. Analyses of growth on individual and mixed carbon substrates were made to determine sugar assimilation patterns in batch and chemostat cultures on coconut water. Growth on the polyol produced a much reduced specific growth rate, assimilation rate, growth yield and productivity compared to growth on the sugars. In mixed substrate fermentations a sequential utilization of the carbohydrates occurred. Both the monosaccharides repressed invertase synthesis and all three sugars repressed sorbitol assimilation. Complete carbon assimilation was only obtained by prolonged batch fermentation or in chemostat cultures at low dilution rates (<0.10 h^-1). Supplementation of coconut water with biotin and nicotinic acid increased biomass yields in chemostat cultures.