Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: possible consequence of energy-dependent lactate export

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.     

Respiratory capacities of mitochondria of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 grown under glucose limitation

A comparative study was made of the in vitro respiratory capacity of mitochondria isolated from Saccharomyces cerevisiae and Candida utilis grown in glucose-limited chemostat cultures. An electron-microscopic analysis of whole cells revealed that the volume density of mitochondria was the same in both yeasts. Mitochondria from both organisms exhibited respiratory control with NADH, pyruvate + malate, 2-oxoglutarate + acetate or malate, and ethanol. The rate of oxidation of these compounds by isolated mitochondria was the same in both yeasts. The rate of oxidation of NADPH by mitochondria from S. cerevisiae was 10 times lower than by those from C. utilis. However, this low rate probably has no influence on the overall in vivo respiratory capacity of S. cerevisiae. The results are discussed in relation to the differences in metabolic behaviour between S. cerevisiae and C. utilis upon transition of cultures from glucose limitation to glucose excess. It is concluded that the occurrence of alcoholic fermentation in S. cerevisiae under these conditions does not result from a bottleneck in the respiratory capacity of the mitochondria.     

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enzymic analysis of the crabtree effect in glucose-limited chemostat cultures of Saccharomyces cerevisiae.

The physiology of Saccharomyces cerevisiae CBS 8066 was studied in glucose-limited chemostat cultures. Below a dilution rate of 0.30 h-1 glucose was completely respired, and biomass and CO2 were the only products formed. Above this dilution rate acetate and pyruvate appeared in the culture fluid, accompanied by disproportional increases in the rates of oxygen consumption and carbon dioxide production. This enhanced respiratory activity was accompanied by a drop in cell yield from 0.50 to 0.47 g (dry weight) g of glucose-1. At a dilution rate of 0.38 h-1 the culture reached its maximal oxidation capacity of 12 mmol of O2 g (dry weight)-1 h-1. A further increase in the dilution rate resulted in aerobic alcoholic fermentation in addition to respiration, accompanied by an additional decrease in cell yield from 0.47 to 0.16 g (dry weight) g of glucose-1. Since the high respiratory activity of the yeast at intermediary dilution rates would allow for full respiratory metabolism of glucose up to dilution rates close to mumax, we conclude that the occurrence of alcoholic fermentation is not primarily due to a limited respiratory capacity. Rather, organic acids produced by the organism may have an uncoupling effect on its respiration. As a result the respiratory activity is enhanced and reaches its maximum at a dilution rate of 0.38 h-1. An attempt was made to interpret the dilution rate-dependent formation of ethanol and acetate in glucose-limited chemostat cultures of S. cerevisiae CBS 8066 as an effect of overflow metabolism at the pyruvate level. Therefore, the activities of pyruvate decarboxylase, NAD+- and NADP+-dependent acetaldehyde dehydrogenases, acetyl coenzyme A (acetyl-CoA) synthetase, and alcohol dehydrogenase were determined in extracts of cells grown at various dilution rates. From the enzyme profiles, substrate affinities, and calculated intracellular pyruvate concentrations, the following conclusions were drawn with respect to product formation of cells growing under glucose limitation. (i) Pyruvate decarboxylase, the key enzyme of alcoholic fermentation, probably already is operative under conditions in which alcoholic fermentation is absent. The acetaldehyde produced by the enzyme is then oxidized via acetaldehyde dehydrogenases and acetyl-CoA synthetase. The acetyl-CoA thus formed is further oxidized in the mitochondria. (ii) Acetate formation results from insufficient activity of acetyl-CoA synthetase, required for the complete oxidation of acetate. Ethanol formation results from insufficient activity of acetaldehyde dehydrogenases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Competition for glucose between the yeasts Saccharomyces cerevisiae and Candida utilis.

The competition between the yeasts Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 for glucose was studied in sugar-limited chemostat cultures. Under aerobic conditions, C. utilis always successfully completed against S. cerevisiae. Only under anaerobic conditions did S. cerevisiae become the dominant species. The rationale behind these observations probably is that under aerobic glucose-limited conditions, high-affinity glucose/proton symporters are present in C. utilis, whereas in S. cerevisiae, glucose transport occurs via facilitated diffusion with low-affinity carriers. Our results explain the frequent occurrence of infections by Crabtree-negative yeasts during bakers' yeast production.     

Homofermentative Lactate Production Cannot Sustain Anaerobic Growth of Engineered Saccharomyces cerevisiae: Possible Consequence of Energy-Dependent Lactate Export

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.     

By-product formation during exposure of respiring Saccharomyces cerevisiae cultures to excess glucose is not caused by a limited capacity of pyruvate carboxylase

Upon exposure to excess glucose, respiring cultures of Saccharomyces cerevisiae produce substantial amounts of ethanol and acetate. A possible role of a limited anaplerotic capacity in this process was investigated by overexpressing pyruvate carboxylase and by replacing it with a heterologous enzyme (Escherichia coli phosphoenolpyruvate carboxylase). Compared to the wild-type, neither the pyruvate carboxylase (Pyc)-overexpressing nor the transgenic strain exhibited reduced by-product formation after glucose pulses to aerobic glucose-limited chemostat cultures. An increased intracellular malate concentration was observed in the two engineered strains. It is concluded that by-product formation in S. cerevisiae is not caused by a limited anaplerotic capacity.     

Transient-State Analysis of Metabolic Fluxes in Crabtree-Positive and Crabtree-Negative Yeasts

In bakers' yeast, an immediate alcoholic fermentation begins when a glucose pulse is added to glucose-limited, aerobically grown cells. The mechanism of this short-term Crabtree effect was investigated via a comparative enzymic analysis of eight yeast species. It was established that the fermentation rate of the organisms upon transition from glucose limitation to glucose excess is positively correlated with the level of pyruvate decarboxylase (EC 4.1.1.1). In the Crabtree-negative yeasts, the pyruvate decarboxylase activity was low and did not increase when excess glucose was added. In contrast, in the Crabtree-positive yeasts, the activity of this enzyme was on the average sixfold higher and increased after exposure to glucose excess. In Crabtree-negative species, relatively high activities of acetaldehyde dehydrogenases (EC 1.2.1.4 and EC 1.2.1.5) and acetyl coenzyme A synthetase (EC 6.2.1.1), in addition to low pyruvate decarboxylase activities, were present. Thus, in these yeasts, acetaldehyde can be effectively oxidized via a bypass that circumvents the reduction of acetaldehyde to ethanol. Growth rates of most Crabtree-positive yeasts did not increase upon transition from glucose limitation to glucose excess. In contrast, the Crabtree-negative yeasts exhibited enhanced rates of biomass production which in most cases could be ascribed to the intracellular accumulation of reserve carbohydrates. Generally, the glucose consumption rate after a glucose pulse was higher in the Crabtree-positive yeasts than in the Crabtree-negative yeasts. However, the respiratory capacities of steady-state cultures of Crabtree-positive yeasts were not significantly different from those of Crabtree-negative yeasts. Thus, a limited respiratory capacity is not the primary cause of the Crabtree effect in yeasts. Instead, the difference between Crabtree-positive and Crabtree-negative yeasts is attributed to differences in the kinetics of glucose uptake, synthesis of reserve carbohydrates, and pyruvate metabolism.     

A biochemical explanation for lipid accumulation in Candida 107 and other oleaginous micro-organisms.

The biochemical explanation for lipid accumulation was investigated principally in Candida 107 and, for comparison, in the non-oleaginous yeast Candida utilis. There were no significant differences between these two yeasts in their control of glucose uptake; in both yeasts, the rates of glucose uptake were independent of the growth rate and were higher in carbon-limited chemostat cultures than in nitrogen-limited cultures. There was no lipid turnover in either yeast, as judged from [14C]acetate uptake and subsequent loss of 14C from the lipid of steady-state chemostat cultures. Acetyl-CoA carboxylase from both yeasts was similar in most characteristics except that from Candida 107 was activated by citrate (40% activation at 1 mM). The enzyme from Candida 107 was relatively unstable and, when isolated from nitrogen-limited (lipid-accumulating) cultures, was accompanied by a low molecular weight inhibitor. The reason for lipid accumulation is attributed to the decrease in the intracellular concentration of AMP as cultures become depleted of nitrogen. As the NAD+-dependent isocitrate dehydrogenase of Candida 107, but not C. utilis, requires AMP for activity, the metabolism of citrate through the tricarboxylic acid cycle in the mitochondria becomes arrested. In Candida 107, but not in C. utilis, there is an active ATP:citrate lyase which converts the accumulating citrate, when it passes into the cytosol, into acetyl-CoA and oxaloacetate. The former product is then available for fatty acid biosynthesis which is stimulated by the high ATP concentration within the cells, by the activation of acetyl-CoA carboxylase by citrate and by the provision of NADPH generated as oxaloacetate is converted via malate to pyruvate. Similar characteristics were evident in oleaginous strains of Rhodotorula glutinis and Mucor circinelloides but not in non-oleaginous representatives of these species.     

Steady-state and transient-state analysis of growth and metabolite production in a Saccharomyces cerevisiae strain with reduced pyruvate-decarboxylase activity

Pyruvate decarboxylase is a key enzyme in the production of low-molecular-weight byproducts (ethanol, acetate) in biomass-directed applications of Saccharomyces cerevisiae. To investigate whether decreased expression levels of pyruvate decarboxylase can reduce byproduct formation, the PDC2 gene, which encodes a positive regulator of pyruvate-decarboxylase synthesis, was inactivated in the prototrophic strain S. cerevisiae CEN. PK113-7D. This caused a 3-4-fold reduction of pyruvate-decarboxylase activity in glucose-limited, aerobic chemostat cultures grown at a dilution rate of 0.10 h(-1). Upon exposure of such cultures to a 50 mM glucose pulse, ethanol and acetate were the major byproducts formed by the wild type. In the pdc2Delta strain, formation of ethanol and acetate was reduced by 60-70%. In contrast to the wild type, the pdc2Delta strain produced substantial amounts of pyruvate after a glucose pulse. Nevertheless, its overall byproduct formation was ca. 50% lower. The specific rate of glucose consumption after a glucose pulse to pdc2Delta cultures was about 40% lower than in wild-type cultures. This suggests that, at reduced pyruvate-decarboxylase activities, glycolytic flux is controlled by NADH reoxidation. In aerobic, glucose-limited chemostat cultures, the wild type exhibited a mixed respiro-fermentative metabolism at dilution rates above 0.30 h(-1). Below this dilution rate, sugar metabolism was respiratory. At dilution rates up to 0.20 h(-1), growth of the pdc2Delta strain was respiratory and biomass yields were similar to those of wild-type cultures. Above this dilution rate, washout occurred. The low micro(max) of the pdc2Delta strain in glucose-limited chemostat cultures indicates that occurrence of respiro-fermentative metabolism in wild-type cultures is not solely caused by competition of respiration and fermentation for pyruvate. Furthermore, it implies that inactivation of PDC2 is not a viable option for reducing byproduct formation in industrial fermentations.     

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.     

Glucose transport in crabtree-positive and crabtree-negative yeasts

The kinetic parameters of glucose transport in four Crabtree-positive and four Crabtree-negative yeasts were determined. The organisms were grown in aerobic glucose-limited chemostats at a dilution rate of 0.1 h-1. The results show a clear correlation between the presence of high-affinity glucose transport systems and the absence of aerobic fermentation upon addition of excess glucose to steady-state cultures. The presence of these H+-symport systems could be established by determination of intracellular accumulation of 6-deoxy-[3H]glucose and alkalinization of buffered cell suspensions upon addition of glucose. In contrast, the yeasts that did show aerobic alcoholic fermentation during these glucose pulse experiments had low-affinity facilitated-diffusion carriers only. In the yeasts examined the capacity of the glucose transport carriers was higher than the actual glucose consumption rates during the glucose pulse experiments. The relationship between the rate of sugar consumption and the rate of alcoholic fermentation was studied in detail with Saccharomyces cerevisiae. When S. cerevisiae was pulsed with low amounts of glucose or mannose, in order to obtain submaximal sugar consumption rates, fermentation was already occurring at sugar consumption rates just above those which were maintained in the glucose-limited steady-state culture. The results are interpreted in relation with the Crabtree effect. In Crabtree-positive yeasts, an increase in the external glucose concentration may lead to unrestricted glucose uptake by facilitated diffusion and hence, to aerobic fermentation. In contrast, Crabtree-negative yeasts may restrict the entry of glucose by their regulated H+-symport systems and thus prevent the occurrence of overflow metabolism.     

Physiological characterization and fed-batch production of an extracellular maltase of Schizosaccharomyces pombe CBS 356

The fission yeast Schizosaccharomyces pombe CBS 356 exhibits extracellular maltase activity. This activity may be of commercial interest as it exhibited a low pH optimum (3.5) and a high affinity for maltose (Km of 7.0+/-1.8 mM). N-terminal sequencing of the protein indicates that it is the product of the AGL1 gene. Regulation of this gene occurs via a derepression/repression mechanism. In sugar- or nitrogen-limited chemostat cultures, the specific rate of enzyme production (q(p)) was independent of the nature of the carbon source (i.e. glucose or maltose), but synthesis was partially repressed by high sugar concentrations. Furthermore, q(p) increased linearly with specific growth rate (mu) between 0.04 and 0.10 h(-1). The enzyme is easily mass-produced in aerobic glucose-limited fed-batch cultures, in which the specific growth rate is controlled to prevent alcoholic fermentation. In fed-batch cultures in which biomass concentrations of 83 g L(-1) were attained, the enzyme concentration reached 58,000 Units per liter culture supernatant. Extracellular maltase may be used as a dough additive in order to prevent mechanisms such as maltose-induced glucose efflux and maltose-hypersensitivity that occur in maltose-consuming Saccharomyces cerevisiae.     

Metabolic Flux Responses to Pyruvate Kinase Knockout in Escherichia coli

The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13C labeling experiments with [U-13C6]glucose in glucose- or ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.     

Steady-state and transient-state analyses of aerobic fermentation in Saccharomyces kluyveri

Some yeasts, such as Saccharomyces cerevisiae, produce ethanol at fully aerobic conditions, whereas other yeasts, such as Kluyveromyces lactis, do not. In this study we investigated the occurrence of aerobic alcoholic fermentation in the petite-negative yeast Saccharomyces kluyveri that is only distantly related to S. cerevisiae. In aerobic glucose-limited continuous cultures of S. kluyveri, two growth regimens were observed: at dilution rates below 0.5 h(-1) the metabolism was purely respiratory, and at dilution rates above 0.5 h(-1) the metabolism was respiro-fermentative. The dilution rate at which the switch in metabolism occurred, i.e. the critical dilution rate, was 66% higher than the typical critical dilution rate of S. cerevisiae. The maximum specific oxygen consumption rate around the critical dilution rate was found to 13.6 mmol (g dry weight)(-1) h(-1) and the capacity of the pyruvate dehydrogenase-bypass pathway was estimated to be high from in vitro enzyme activities; especially the specific activity of acetyl-CoA synthetase was much higher than in S. cerevisiae at all tested conditions. Addition of glucose to respiring cells of S. kluyveri led to ethanol formation after a delay of 20-50 min (depending on culture conditions prior to the pulse), which is in contrast to S. cerevisiae that ferments immediately after glucose addition.     

Overproduction of threonine aldolase circumvents the biosynthetic role of pyruvate decarboxylase in glucose-limited chemostat cultures of Saccharomyces cerevisiae

Pyruvate decarboxylase-negative (Pdc(-)) mutants of Saccharomyces cerevisiae require small amounts of ethanol or acetate to sustain aerobic, glucose-limited growth. This nutritional requirement has been proposed to originate from (i) a need for cytosolic acetyl coenzyme A (acetyl-CoA) for lipid and lysine biosynthesis and (ii) an inability to export mitochondrial acetyl-CoA to the cytosol. To test this hypothesis and to eliminate the C(2) requirement of Pdc(-) S. cerevisiae, we attempted to introduce an alternative pathway for the synthesis of cytosolic acetyl-CoA. The addition of L-carnitine to growth media did not restore growth of a Pdc(-) strain on glucose, indicating that the C(2) requirement was not solely due to the inability of S. cerevisiae to synthesize this compound. The S. cerevisiae GLY1 gene encodes threonine aldolase (EC 4.1.2.5), which catalyzes the cleavage of threonine to glycine and acetaldehyde. Overexpression of GLY1 enabled a Pdc(-) strain to grow under conditions of carbon limitation in chemostat cultures on glucose as the sole carbon source, indicating that acetaldehyde formed by threonine aldolase served as a precursor for the synthesis of cytosolic acetyl-CoA. Fractionation studies revealed a cytosolic localization of threonine aldolase. The absence of glycine in these cultures indicates that all glycine produced by threonine aldolase was either dissimilated or assimilated. These results confirm the involvement of pyruvate decarboxylase in cytosolic acetyl-CoA synthesis. The Pdc(-) GLY1 overexpressing strain was still glucose sensitive with respect to growth in batch cultivations. Like any other Pdc(-) strain, it failed to grow on excess glucose in batch cultures and excreted pyruvate when transferred from glucose limitation to glucose excess.     

Metabolic flux variation of Saccharomyces cerevisiae cultivated in a multistage continuous stirred tank reactor fermentation environment.

The technique of metabolic flux analysis was implemented to elucidate the flux balancing of Saccharomyces cerevisiae cultivated in a multistage continuous stirred tank reactor fermentation environment. The results showed that the majority of the substrate (97.70 +/- 0.49%) was funneled into the glycolytic pathway, while the remainder was subdivided between the pentose phosphate pathway and pathways for polysaccharide synthesis. At the pyruvate node, 87.30 +/- 1.38% of the flux was channeled through the reaction governed by pyruvate decarboxylase. Fluxes through the pyruvate dehydrogenase bypass were maintained at a constant level (82.65 +/- 1.47%) irrespective of the configuration of the fermentation setup. Activity through the TCA "cycle" was replenished by the reaction catalyzed by pyruvate carboxylase and by the transport of cytosolic oxaloacetate across the mitochondrial membrane. The CO(2) evolution rate varied as fermentation progressed; however, the yield coefficient of CO(2) remained at a constant value. Although a constant yield of ethanol (0.42 g of ethanol/g of glucose) was obtained, operations of the TCA cycle were gradually switched from partially reductive to partially oxidative pathways from the first fermenter to the fourth fermenter.     

Growth Rate-Dependent Modulation of Carbon Flux through Central Metabolism and the Kinetic Consequences for Glucose-Limited Chemostat Cultures of Corynebacterium glutamicum

The physiological behavior of Corynebacterium glutamicum in glucose-limited chemostat cultures was examined from both growth kinetics and enzymatic viewpoints. Metabolic fluxes within the central metabolism were calculated from growth kinetics and analyzed in relation to specific enzyme activities. At high growth rates, incomplete glucose removal was observed, and this was attributed to rate-limiting capacity of the phosphotransferase system transporter and the probable contribution of a low-affinity permease uptake mechanism. The improved biomass yield observed at high growth rates was related to a shift in the profile of anaplerotic carboxylation reactions, with pyruvate carboxylase replacing malic enzyme. Phosphoenolpyruvate carboxylase, an activity often assumed to be the major anaplerotic reaction during growth of C. glutamicum on glucose, was present at only low levels and is unlikely to contribute significantly to tricarboxylic acid cycle fuelling other than at low growth rates.