The effect of succinate on respiration, transamination, and pyruvate formation in cells of the yeast Dipodascus magnusii

The effect of succinate on the growth and respiration of the yeast Dipodascus magnusii VKM Y-1072, which is auxotrophic for thiamine and biotin, was studied. The addition of succinate to a culture grown on glucose was found to activate the respiration of cells on various substrates by enhancing the processes related to transamination reactions. In this case, aerobic fermentation (ethanol production) decreased, whereas pyruvate production increased. When succinate was added to the medium as the sole carbon source, it supported yeast growth in the absence of one of the two vitamins, thiamine or biotin, but not both. The yeast metabolism was completely respiratory, without any signs of aerobic fermentation. A drastic rise in pyruvate production in the yeast grown on glucose in the presence of succinate and the absence of biotin are also indicative of metabolic changes.     

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.     

碳源对重组大肠杆菌两阶段发酵产丁二酸的影响

研究了在好氧培养基中分别添加不同碳源对两阶段发酵菌体生长、酶活及代谢产物分布的影响,结果表明添加4mmol/L葡萄糖和12,54,80mmol/L乙酸钠均可以提高好氧阶段的菌体密度和相关酶活。将不同条件下培养的菌体转接厌氧发酵后,厌氧阶段的酶活和代谢产物分布也发生改变。进一步对酶活及代谢产物分析表明：Escherichia coli NZN111(sfcA)厌氧发酵过程中,磷酸烯醇式丙酮酸羧化激酶(PCK)是产丁二酸的关键酶,丙酮酸激酶(PYK)主要和副产物丙酮酸的积累有关,异柠檬酸裂解酶(ICL)对丁二酸产量也有一定影响。好氧培养基中添加80mmol/L乙酸钠,厌氧发酵结束时丁二酸的质量收率可达89.0%,相比对照提高了16.6%。     

Effects of glucose, vitamins, and DO concentrations on pyruvate fermentation using Torulopsis glabrata IFO 0005 with metabolic flux analysis

The effects of glucose, vitamins, and DO concentrations on efficient pyruvic acid fermentation were investigated using Torulopsis glabrata IFO 0005, and a novel biphasic culture method was developed on the basis of the metabolic flux analysis. T. glabrata requires the four vitamins nicotinic acid (NA), thiamine hydrochloride (B(1)), pyridoxine hydrochloride, and biotin for cell growth. The deficiency of these vitamins plays an essential role in pyruvate fermentation. In the present study, we considered the effects of the first two vitamins on the pyruvate fermentation. On the basis of several batch and fed-batch experiments, it was found that, as a result of glucose inhibition of cell growth, the initial glucose concentration should be around 30-40 g/L, and the glucose concentration during fermentation should be controlled at high level around 30 g/L. On the basis of an analysis of carbon flux distribution, a biphasic fermentation method was developed where the cultivation started with a high DO (at 40-50% of air saturation) for efficient cell growth and then was reduced to 5-10% for efficient pyruvate production. Since a fair amount of ethanol was formed when the DO concentration was decreased, the addition of NA turned out to be effective in reducing the ethanol formation. This may be due to a relaxing of the requirement for NADH oxidation by the alcohol dehydrogenase pathway. Since B(1) affects both the pyruvate dehydrogenase complex and pyruvate decarboxylase, its initial concentration must be carefully determined by considering both the cell growth and pyruvate production phases.     

Synthesis of Mono- and Sesquiterpenoids

The synthesis of isocitrate lyase in Candida tropicalis, the growth of which was stimulated by exogenously added biotin, was released from repression by glucose under biotin-deficient conditions. Biotin deficiency reduced remarkably the levels of biotin-enzymes, pyruvate carboxylase and acetyl-Co A carboxylase, in the glucose-utilizing cells of this yeast. A marked increase in intracellular level of pyruvate was observed in the biotin-deficient cells. Acetyl-CoA-donating compounds, such as pyruvate, acetate and alkanes, stimulated the formation of isocitrate lyase in the yeast regardless of the presence or absence of biotin. On the other hand, malate and succinate did not affect the enzyme synthesis. The isocitrate lyase synthesis under biotin-sufficient conditions was repressed by not only glucose but also glucosamine and 2-deoxyglucose. This repression by glucose was not eliminated by cAMP. The stimulated synthesis of isocitrate lyase under biotin-deficient conditions was also observed in C. albicans and C. guilliermondii growing on glucose.     

EFFECTS OF CERTAIN LIMITING CONDITIONS ON THE SYNTHESIS OF B VITAMINS BY YEAST

In yeast crops which were grown in the presence of various inhibitors, there was considerable variation in content of the various B vitamins. A higher degree of parallelism in variation in content was found to exist between thiamine and niacin than between any other pair of vitamins; this has been interpreted as indicating that the predominant functions of these two vitamins are their established rôles in fermentation. Values for inositol indicate that it may be involved in fermentation processes, but this is not the case for other members of the B complex. Biotin appears to be unique since in no case did the biotin content of yeast grown in the presence of an inhibitor fall below that of the control yeast. There was some evidence of synthesis of biotin, or a material with biotin activity, in the presence of certain inhibitors, the most striking instance being with sulfaguanidine. An exogenous supply of biotin was essential for extensive proliferation of F. B. yeast, and yeast grown in a medium to which biotin was the only added vitamin contained the B vitamins in amounts very similar to those found in the control yeast, the most marked differences being in increased vitamin B₆ and p-aminobenzoic acid contents. In the absence of biotin, significant amounts of all of the B vitamins except biotin were synthesized, both in the presence and absence of certain other members of the B complex. The addition of thiamine, pyridoxine, inositol, and β-alanine to the culture medium caused a reduction in the amounts of vitamin B₆ and p-aminobenzoic acid synthesized. F. B. yeast was able to grow in a xylose medium only when certain of the B vitamins were present, and even then growth was limited. Evidence was obtained for some synthesis of all of the vitamins investigated except biotin and vitamin B₆. The most significant differences in vitamin content between galac yeast and the parent F. B. strain were in folic acid and vitamin B₆, the former being considerably reduced in amount, the latter being increased.     

Oxygen- and glucose-dependent regulation of central carbon metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose(-1)) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.     

Genetic reconstruction of the aerobic central metabolism in Escherichia coli for the absolute aerobic production of succinate

Most reported efforts to enhance production of the industrially valuable specialty chemical succinate have been done under anaerobic conditions, where E. coli undergoes mixed-acid fermentation. These efforts have often been hampered by the limitations of NADH availability, poor cell growth, and slow production. An aerobic succinate production system was strategically designed that allows E. coli to produce and accumulate succinate efficiently and substantially as a product under absolute aerobic conditions. Mutations in the tricarboxylic acid cycle (sdhAB, icd, iclR) and acetate pathways (poxB, ackA-pta) of E. coli were created to construct the glyoxylate cycle for aerobic succinate production. Experiments in flask studies showed that 14.28 mM of succinate could be produced aerobically with a yield of 0.344 mole/mole using 55 mM glucose. In aerobic batch reactor studies, succinate production rate was faster, reaching 0.5 mole/mole in 24 h with a concentration of 22.12 mM; further cultivation showed that succinate production reached 43 mM with a yield of 0.7. There was also substantial pyruvate and TCA cycle C(6) intermediate accumulation in the mutant. The results suggest that more metabolic engineering improvements can be made to this system to make aerobic succinate production more efficient. Nevertheless, this aerobic succinate production system provides the first platform for enhancing succinate production aerobically in E. coli based on the creation of a new aerobic central metabolic network.     

Screening of pyruvate-producing yeast and effect of nutritional conditions on pyruvate production

AIMS: To find a yeast strain that can overproduce pyruvate and to investigate the effect of nutrients on pyruvate production. METHODS AND RESULTS: Trichosporon cutaneum PD70, a yeast strain that can overproduce pyruvate, was isolated from shake-flask cultures of 132 yeast strains. Pyruvate was measured by the HPLC or DNP method (see Materials and methods). Pyruvate production reached approximately 30.0 +/- 1.0 g l(-1) in basal fermentation medium. Different nutrient supplements had great effects on pyruvate production. Some of the conditions that gave the highest yield are described. CONCLUSIONS: Exogenous thiamine supplement caused a decrease in pyruvate yield. Some amino acids, such as L-arginine, L-isoleucine and L-valine, caused a minor increase in pyruvate yield. Soybean peptone was the most suitable nitrogen source for pyruvate production. A glucose concentration of 15% in fermentation medium gave the highest yield (34.6 g l(-1)) and the highest yield against consumed glucose (0.429 g g(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: Nutrients have significant impacts on pyruvate production. As a pyruvate overproducing yeast strain independent of exogenous vitamins or amino acids, T. cutaneum PD70 provides an advantage for commercial pyruvate production.     

Microcalorimetric investigations of the metabolism of yeasts VII. Flow-calorimetry of aerobic batch cultures

Summary The heat evolution of aerobic batch cultures of growing yeast (Saccharomyces cerevisiae) in glucose media was investigated by a combination of a flow-microcalorimeter with a fermentor vessel. The course of heat production, cell production and the rate of oxygen consumption were qualitatively the same for all glucose concentrations between 10 mM and 100 mM. Under optimal aerobic conditions a triphasic growth was observed due to the fermentation of glucose to ethanol, respiration of ethanol to CO₂ and acetate, and respiration of acetate to C0₂. Energy and carbon were found to be in balance for all glucose concentrations.     

Respiratory metabolism of a "petite negative"yeast Schizosaccharomyces pombe 972h-

The respiratory metabolism of Schizosaccharomyces pombe 972h(-), a fission, haplontic, "petite negative" yeast, was studied. Glucose and glycerol are good growth substrates and are oxidized under appropriate conditions. l-Lactate, ethanol, malate, and succinate are oxidized but are poor substrates for growth. d-Lactate and pyruvate are neither oxidized nor used for growth. Limited growth was observed under anaerobic conditions. The addition of 0.3% KNO(3) to a rich medium relieves the oxygen requirement. A continuous increase of cell respiration during growth on repressive concentration of glucose was observed, suggesting the presence of glucose repression of respiration. Reduced nicotinamide adenine dinucleotide (NADH), succinate, alpha-glycerophosphate, and ascorbate plus tetramethyl-p-phenylenediamine are oxidized by a mitochondrial fraction. NADH and succinate oxidations are inhibited by antimycin A and NaCN but not by rotenone, suggesting the absence of the phosphorylation site I and the presence of sites II and III. The effects of several mitochondrial inhibitors on growth and respiration indicate that the requirement of an oxidant for growth is related neither to the functioning of the respiratory electron transport chain nor to the formation of respiratory energy. The previously suggested correlations between the nonviability of vegetative "petites" mutants, the absence of repression of respiration by glucose, and the incapacity to grow under anaerobic conditions are thus not strictly valid for S. pombe.     

Oxygen- and Glucose-Dependent Regulation of Central Carbon Metabolism in Pichia anomala

We investigated the regulation of the central aerobic and hypoxic metabolism of the biocontrol and non-Saccharomyces wine yeast Pichia anomala. In aerobic batch culture, P. anomala grows in the respiratory mode with a high biomass yield (0.59 g [dry weight] of cells g of glucose⁻¹) and marginal ethanol, glycerol, acetate, and ethyl acetate production. Oxygen limitation, but not glucose pulse, induced fermentation with substantial ethanol production and 10-fold-increased ethyl acetate production. Despite low or absent ethanol formation, the activities of pyruvate decarboxylase and alcohol dehydrogenase were high during aerobic growth on glucose or succinate. No activation of these enzyme activities was observed after a glucose pulse. However, after the shift to oxygen limitation, both enzymes were activated threefold. Metabolic flux analysis revealed that the tricarboxylic acid pathway operates as a cycle during aerobic batch culture and as a two-branched pathway under oxygen limitation. Glucose catabolism through the pentose phosphate pathway was lower during oxygen limitation than under aerobic growth. Overall, our results demonstrate that P. anomala exhibits a Pasteur effect and not a Crabtree effect, i.e., oxygen availability, but not glucose concentration, is the main stimulus for the regulation of the central carbon metabolism.     

Pyruvate flux distribution in NADH-oxidase-overproducing Lactococcus lactis strain as a function of culture conditions

The influence of growth conditions on product formation from glucose by Lactococcus lactis strain NZ9800 engineered for NADH-oxidase overproduction was examined. In aerobic batch cultures, a large production of acetoin and diacetyl was found at acidic pH under pH-unregulated conditions. However, pyruvate flux was mainly driven towards lactate production when these cells were grown under strictly pH-controlled conditions. A decreased NADH-oxidase overproduction accompanied the homolactic fermentation, suggesting that the cellular energy was used with preference to maintain cellular homeostasis rather than for NADH-oxidase overproduction. The end product formation and NADH-oxidase activity were also studied in cells grown in aerobic continuous cultures under acidic conditions. A homoacetic type of fermentation as well as a low NADH-oxidase overproduction were observed at low dilution rates. NADH-oxidase was efficiently overproduced as the dilution rate was increased and consequently metabolic flux through lactate dehydrogenase drastically decreased. Under these conditions the flux limitation via pyruvate dehydrogenase was relieved and this enzymatic complex accommodated most of the pyruvate flux. Pyruvate was also significantly converted to acetoin and diacetyl via alpha-acetolactate synthase. At higher dilution rates, acetate production declined and the cultures turned to mixed-acid fermentation. These results suggest that the need to maintain the cellular homeostasis influenced NADH-oxidase overproduction and consequently the end product formation from glucose in these engineered strains.     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

The vitamin requirements of Candida shehatae for xylose fermentation

Abstract The vitamin requirements of a strain of Candida shehatae for the fermentation of d -xylose was determined using a statistical procedure with a 2³ factorial design. Biotin as well as thiamine exerted a dramatic stimulatory effect on the rate of ethanol production, coupled with a significant improvement in the ethanol yield. The greatest enhancement of the fermentation was found in the presence of both these vitamins. Pyridoxine exerted only a minor effect, but was essential for complete substrate utilization in the absence of either biotin or thiamine. Only biotin caused a significant increase in the growth rate.     

Production of B-group vitamins by two Rhizobium strains in chemically defined media

Twenty-eight strains of Rhizobium spp. were tested for their ability to grow in chemically-defined medium lacking growth factors. Two strains, R. meliloti GR4B and Rhizobium spp. ( Acacia ) GRH28, were selected, on the basis of their good growth under the conditions imposed, for further quantification of the production of water-soluble vitamins (thiamine, niacin, riboflavin, pantothenic acid and biotin) in chemically defined media amended with different compounds (mannitol, glucose or sodium succinate) as sole carbon sources. Qualitative and quantitative production of vitamins in chemically-defined media was significantly affected by the use of C sources of a different nature and the age of the cultures. Strain GRH28 produced all the vitamins analysed, and high biological levels of biotin (14 ng ml^–1 culture) were detected after 6 d of culture in mineral medium amended with mannitol. Pantothenic acid was the vitamin detected in the highest amounts (up to 1 μg ml^–1 of culture) in culture supernatant fluids of strain GR4B grown for 6 d with succinate as sole carbon source.     

Regulation of reductive production of succinate under anaerobic conditions in baker's yeast

When baker's yeast grown aerobically on ethanol as a carbon source was anaerobically cultured in a medium containing glucose, the activity of a cytoplasmic fumarate reductase irreversibly catalyzing the conversion of fumarate to succinate increased, reaching about 3 times the original activity after 12 h, while the activity of succinate dehydrogenase was almost lost after 10 h. These results indicate that the citrate cycle is partially modified to become a reductive pathway leading to succinate during the anaerobic cultivation. In non-proliferating cells grown anaerobically on glucose, the rates of accumulating succinate and pyruvate were decreased and increased, respectively, with increasing concentrations of L-aspartate or NH4Cl in the medium containing glucose as a substrate. These changes were accompanied with increase in the cellular content of aspartate, an inhibitor of pyruvate carboxylase that is involved in supplying the intermediates of the citrate cycle, and pyruvate, a substrate of the enzyme. The aminotransferase inhibitor, aminooxyacetate, prevented the changes in succinate accumulation and cellular aspartate following the addition of NH4Cl. The addition of L-glutamate caused a marked increase in the rate of succinate accumulation without changing the cellular content of aspartate. Neither L-glutamate nor L-aspartate had the ability to produce succinate. The rate of glucose consumption was not changed upon adding these nitrogen compounds. Similar findings were also observed in experiments using proliferating cells. This report presents evidence that in cells containing a large amount of the fumarate reductase, the production of succinate from glucose is regulated by the cellular level of aspartate through the pyruvate carboxylase reaction and that glutamate regulates the succinate production by a mechanism distinct from that involved in the regulation by L-aspartate.     

Efficient pyruvate production by a multi-vitamin auxotroph of Torulopsis glabrata: key role and optimization of vitamin levels

A multi-vitamin auxotroph, Torulopsis glabrata strain WSH-IP303, which can use ammonium chloride as a sole nitrogen source for pyruvate production, was selected. To optimize pyruvate yield and productivity, a simple but useful, orthogonal design method, was used to investigate the relationship between thiamine, nicotinic acid, pyridoxine, biotin, and riboflavin. Thiamine was confirmed to be the most important factor affecting pyruvate production. When the concentration of thiamine was 0.01 mg/l or 0.015 mg/l, glucose consumption was improved by increasing the nicotinic acid concentration. When the concentrations of nicotinic acid, thiamine, pyridoxine, biotin, and riboflavin were 8.0, 0.015, 0.4, 0.04, and 0.1 mg/l, respectively, pyruvate concentration and yield reached 52 g/l and 0.52 g/g, respectively, in a 48-h flask culture. By employing a combination of the optimum vitamin concentrations, a batch culture was conducted in a 2.5-l fermentor with an initial glucose concentration of 112 g/l; and the pyruvate concentration reached 69 g/l after 56 h (yielding 0.62 g/g).     

Characterization of aerobic and anaerobic vegetative growth of the food-borne pathogen Bacillus cereus F4430/73 strain

The Gram-positive bacterium Bacillus cereus is a facultative anaerobe that is still poorly characterized metabolically. In this study, the aerobic vegetative growth and anaerobic vegetative growth of the food-borne pathogen B. cereus F4430/73 strain were compared with those of the genome-sequenced ATCC14579 strain using glucose and glycerol as fermentative and nonfermentative carbon sources, respectively. Uncontrolled batch cultures on several defined media showed that B. cereus strains had high amino acid or pyruvate requirements for anaerobic fermentative growth. In addition, growth performance was considerably improved by maintaining the pH of the culture medium near neutrality. Spectra of fermentation by-products were typically (per mole of glucose) 0.2-0.4 acetate, 1.1-1.4 L-lactate, 0.3-0.4 formate, and 0.05-0.2 ethanol with only traces of succinate, pyruvate, and 2,3-butanediol. These spectra were drastically changed in the presence of 20 mmol nitrate x L(-1), which stimulated anaerobic growth. During anaerobic and aerobic respiration, the persistent production of acetate and other by-products indicated overflow metabolisms. This was especially true in glucose-grown cells for which respiratory complex III made only a minor contribution to growth. Surprisingly, oxygen uptake rates linked to the cytochrome c and quinol branches of the respiratory chain were maintained at high levels in anaerobic, respiring, or fermenting cells. Growth and metabolic features of B. cereus F4430/73 are discussed using biochemical and genomic data.