Stoichiometry, kinetics, and regulation of glucose and amino acid metabolism of a recombinant BHK cell line in batch and continuous cultures

Batch and continuous cultures were carried out to study the stoichiometry, kinetics, and regulation of glucose and amino acid metabolism of a recombinant BHK cell line, with particular attention to the metabolism at low levels of glucose and glutamine. The apparent yields of cells on glucose and glutamine, lactate on glucose, and ammonium on glutamine were all found to change significantly at low residual concentrations of glucose (< 5 mmol/L) and glutamine (< 1 mmol/L). The uptake rates of glucose and glutamine were markedly reduced at low concentrations, leading to a more effective utilization of these nutrients for energy metabolism and biosynthesis and reduced formation rates of lactate and ammonium. However, the consumption of other amino acids, especially the essential amino acids leucine, isoleucine, and valine and the nonessential amino acids serine and glutamate, was strongly enhanced at low glutamine concentration. Quantitatively, it was shown that the cellular yields and rates associated with glucose metabolism were primarily determined by the residual glucose concentration, while those associated with glutamine metabolism depended mainly on the residual glutamine. Both experimental results and analysis of the kinetic data with models showed that the glucose metabolism of BHK cells is not affected by glutamine except for a slight influence under glucose limitation and glutaminolysis not by glucose, at least not significantly under the experimental conditions. Compared to hybridoma and other cultured animal cells, the recombinant BHK cell line showed remarkable differences in terms of nutrient sensitivity, stoichiometry, and amino acid metabolism at low levels of nutrients. These cell-line-specific stoichiometry and nutrient needs should be considered when designing an optimal medium and/or feeding strategy for achieving high cell density and high productivity of BHK cells. In this work, a cell density of 1.1 x 10(7) cells/mL was achieved in a conventional continuous culture by using a proper feed medium.     

Glucocorticoids increase retinoid-X receptor alpha (RXRalpha) expression and enhance thyroid hormone action in primary cultured rat hepatocytes

A dynamic model of glucose overflow metabolism in batch and fed-batch cultivations of Escherichia coli W3110 under fully aerobic conditions is presented. Simulation based on the model describes cell growth, respiration, and acetate formation as well as acetate reconsumption during batch cultures, the transition of batch to fed-batch culture, and fed-batch cultures. E. coli excreted acetate only when specific glucose uptake exceeded a critical rate corresponding to a maximum respiration rate. In batch cultures where the glucose uptake was unlimited, the overflow acetate made up to 9. 0 +/- 1.0% carbon/carbon of the glucose consumed. The applicability of the model to dynamic situations was tested by challenging the model with glucose and acetate pulses added during the fed-batch part of the cultures. In the presence of a glucose feed, E. coli utilized acetate 3 times faster than in the absence of glucose. The cells showed no significant difference in maximum specific uptake rate of endogenous acetate produced by glucose overflow and exogenous acetate added to the culture, the value being 0.12-0.18 g g-1 h-1 during the entire fed-batch culture period. Acetate inhibited the specific growth rate according to a noncompetitive model, with the inhibition constant (ki) being 9 g of acetate/L. This was due to the reduced rate of glucose uptake rather than the reduced yield of biomass.     

Sequence of the gene encoding a highly thermostable neutral proteinase from Bacillus sp. strain EA1: expression in Escherichia coli and characterisation

Ammonium assimilation was studied by feeding [15N]ammonium to actively growing mycelium of Agaricus bisporus. Products of ammonium assimilation were analysed using 15N-NMR. Participation of glutamine synthetase, glutamate synthase and NADP-dependent glutamate dehydrogenase was determined by inhibiting glutamine synthetase with phosphinothricin and glutamate synthase with azaserine. Our results clearly indicate that, under the conditions used, ammonium assimilation is mainly catalysed by the enzymes of the glutamine synthetase/glutamate synthase pathway. No indications were found for participation of NADP-dependent glutamate dehydrogenase. Furthermore, 15N-labelling shows that transamination of glutamate with pyruvate to yield alanine is a major route in nitrogen metabolism. Another major route is the formation of N-acetylglucosamine. Compared to the formation of N-acetylglucosamine there was only a limited formation of arginine.     

A nutrient-regulated cytosolic calcium oscillator in endocrine pancreatic glucagon-secreting cells

We investigated the influence of nutrients on spontaneous cytosolic calcium oscillations in InR1-G9 glucagonoma cells, a model for pancreatic alpha-cells. The oscillations depended on calcium release from stores and on calcium influx, partly through voltage-dependent calcium channels. Oscillations required the presence of at least 1 mM glucose, 50 microM alanine, or 50 microM glutamine, but were terminated by higher nutrient concentrations (40 mM glucose, or above 2 mM alanine or glutamine). The effects depended on the metabolism of the nutrients. Glutamine and alanine hyperpolarized the cells. This effect was inhibited (glutamine) or attenuated (alanine) by 1 mM ouabain. Our findings suggest that [Ca2+]i regulation in alpha-cells is dominated by slow oscillations induced by a lack of metabolic energy, resulting in decreased calcium export and storage, as well as increased calcium influx, partly due to depolarization caused by reduced sodium pump activity. These processes, leading to an elevated cytosolic calcium concentration, may mediate oscillations by calcium-induced calcium release from intracellular stores.     

Influence of the nitrogen source on Saccharomyces cerevisiae anaerobic growth and product formation

To prevent the loss of raw material in ethanol production by anaerobic yeast cultures, glycerol formation has to be reduced. In theory, this may be done by providing the yeast with amino acids, since the de novo cell synthesis of amino acids from glucose and ammonia gives rise to a surplus of NADH, which has to be reoxidized by the formation of glycerol. An industrial strain of Saccharomyces cerevisiae was cultivated in batch cultures with different nitrogen sources, i.e., ammonium salt, glutamic acid, and a mixture of amino acids, with 20 g of glucose per liter as the carbon and energy source. The effects of the nitrogen source on metabolite formation, growth, and cell composition were measured. The glycerol yields obtained with glutamic acid (0.17 mol/mol of glucose) or with the mixture of amino acids (0.10 mol/mol) as a nitrogen source were clearly lower than those for ammonium-grown cultures (0.21 mol/mol). In addition, the ethanol yield increased for growth on both glutamic acid (by 9%) and the mixture of amino acids (by 14%). Glutamic acid has a large influence on the formation of products; the production of, for example, alpha-ketoglutaric acid, succinic acid, and acetic acid, increased compared with their production with the other nitrogen sources. Cultures grown on amino acids have a higher specific growth rate (0.52 h-1) than cultures of both ammonium-grown (0.45 h-1) and glutamic acid-grown (0.33 h-1) cells. Although the product yields differed, similar compositions of the cells were attained. The NADH produced in the amino acid, RNA, and extracellular metabolite syntheses was calculated together with the corresponding glycerol formation. The lower-range values of the theoretically calculated yields of glycerol were in good agreement with the experimental yields, which may indicate that the regulation of metabolism succeeds in the most efficient balancing of the redox potential.     

Effects of Salmonella typhimurium infection and ofloxacin treatment on glucose and glutamine metabolism in Caco-2/TC-7 cells

The effects of both Salmonella typhimurium infection and 5 mM ofloxacin treatment on 2 mM glutamine and 5 mM glucose metabolism in the enterocyte-like Caco-2/TC-7 cell line were studied. These cells utilized glutamine (212.07 +/- 16.75 [mean +/- standard deviation] nmol per h per 10(6) viable cells) and, to a lesser extent, glucose (139.63 +/- 11.52 nmol per h per 10(6) viable cells). Metabolism of these substrates in Caco-2/TC-7 cells resembled that in rat, pig, or human enterocytes. Infection by S. typhimurium C53-enhanced glucose and glutamine substrate utilization by 32 and 22%, respectively and enhanced glucose and glutamine substrate oxidation by eight- and twofold, respectively. These increases in glucose and glutamine metabolism (especially glucose metabolism) were due in part to the metabolism of intracellular bacteria and/or to the activation of cellular metabolism. Substrate metabolism (especially glucose metabolism) in C53-infected cells was partially reduced by treatment with ofloxacin. It was concluded that cellular fuel metabolism is stimulated at the earliest stage of infection (3 to 4 h) and that treatment with 5 mM ofloxacin does not completely restore substrate metabolism to the levels observed in uninfected cells, possibly because this treatment does not eradicate intracellular S. typhimurium completely.     

The effects of 2-deoxyglucose and amino-oxyacetic acid on the radiation response of mammalian cells in vitro

Cells use substrates such as glucose and glutamine to provide energy for repair of radiation damage. Glutaminolysis and glycolysis were inhibited by aminooxyacetic acid (AOAA) and 2-deoxyglucose (2DG), respectively, to inhibit metabolism of these substrates in order to determine the effect on radiation response of CHO-K1 cells in vitro. Exposure to treatments which inhibit energy metabolism resulted in alterations in radiosensitivity and, in general, a reduction in cellular recovery rate after y-irradiation but varied with regard to the extent of recovery. The greatest inhibition of recovery relative to that in normal culture medium was found with medium which lacked glucose and glutamine and contained 2DG and AOAA. In contrast, medium lacking glucose and glutamine without the addition of inhibitors resulted in an increase in recovery. It is proposed that the efficiency of energy pathways such as glycolysis and glutaminolysis and their interaction are determinants of both radiosensitivity and recovery.     

Adaptive alterations in cellular metabolism with malignant transformation

OBJECTIVE: The authors studied the differences between glutamine and glucose utilization in normal fibroblasts and in fibrosarcoma cells to gain insights into the metabolic changes that may occur during malignant transformation. SUMMARY BACKGROUND DATA: The process of malignant transformation requires that cells acquire and use nutrients efficiently for energy, protein synthesis, and cell division. The two major sources of energy for cancer cells are glucose and glutamine. Glutamine is also essential for protein and DNA biosynthesis. We studied glucose and glutamine metabolism in normal and malignant fibroblasts. METHODS: Studies were done in normal rat kidney fibroblasts and in rat fibrosarcoma cells. We measured glutamine transport across the cell membrane, breakdown of glutamine by the enzyme glutaminase (the first step in oxidation), glutamine and glucose oxidation rates to CO2, rates of protein synthesis from glutamine, and glutamine-dependent growth rates. RESULTS: Glutamine transport rates were increased more than sixfold in fibrosarcomas compared to normal fibroblasts. In fibroblasts, glutamine transport was mediated by systems ASC and A. In malignant fibrosarcomas, only system ASC was identifiable, and its Vmax was 15 times higher than that observed in fibroblasts. Despite an increase in transport, glutaminase activity was diminished and glutamine oxidation to CO2 was reduced in fibrosarcomas versus normal fibroblasts. In fibroblasts, glutamine oxidation was 1.8 times higher than glucose oxidation. In contrast, glucose oxidation was 3.5 times greater than glutamine oxidation in fibrosarcomas. Protein synthesis from glutamine transported by fibrosarcomas was threefold greater than that observed in normal fibroblasts. Despite marked increases in glutamine utilization and glucose oxidation in fibrosarcoma cells, growth rates were higher in the normal fibroblasts. CONCLUSIONS: The process of malignant transformation is associated with a marked increase in cellular glutamine transport, which is mediated by a single high-affinity, high-capacity plasma membrane carrier protein. In normal fibroblasts, the transported glutamine is used primarily for energy production via oxidation of glutamine carbons to CO2. In fibrosarcomas, glutamine oxidation falls and glutamine is shunted into protein synthesis; simultaneously, the malignant cell switches to a glucose oxidizer. The increased glutamine transport and glucose oxidation in fibrosarcomas appears to be related to the malignant phenotype and not merely to an increase in cell growth rates.     

Trafficking of molecules and metabolic signals in the retina

Photoreceptors need the support of pigment epithelial (PE) and Müller glial cells in order to maintain visual sensitivity and neurotransmitter resynthesis. In rod outer segments (ROS), all-trans-retinal is transformed to all-trans-retinol by retinol dehydrogenase using NADPH. NADPH is restored in ROS by the pentose phosphate pathway utilizing high amounts of glucose supplied by choriocapillaries. The retinal formed is transported to PE cells where regeneration of 11-cis-retinal occurs. Müller cells take up and metabolize glucose predominantly to lactate which is massively released into the extracellular space (ES). Lactate is taken up by photoreceptors, where it is transformed to pyruvate which, in turn, enters the Krebs cycle in mitochondria of the inner segment. Stimulation of neurotransmitter release by darkness induces 130% rise in the amount of glutamate released into ES. Glutamate is transported into Müller cells where it is predominantly transformed to glutamine. Stimulation of photoreceptors induces an eightfold increase in glutamine formation. It appears, therefore, that there is a signaling function in the transfer of amino acids from Müller cells to photoreceptors. Work on the model-system of the honeybee retina demonstrated that photoreceptors release NH4+ and glutamate in a stimulus-dependent manner which, in turn, contribute to the biosynthesis of alanine in glia. Alanine released into the extracellular space is taken up and used by photoreceptors. Glial cells take glutamate by high-affinity transporters. This uptake induces a transient change in glial cell metabolism. The transformation of glutamate to glutamine is possibly also controlled by the uptake of NH4+ which directly affects cellular metabolism.     

Influence of carbon source on nitrate removal by nitrate-tolerant Klebsiella oxytoca CECT 4460 in batch and chemostat cultures

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h-1, whereas with glycerol it was 0.45 h-1. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (YC) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (YN) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h-1 the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h-1 the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.     

Osteoarthritis involving the metatarsophalangeal joints and management of metatarsophalangeal joint pain via injection therapy

We studied the effect of administration of a mixture of alanine and glutamine on the inhibition of liver regeneration caused by alcohol in rats undergoing partial hepatectomy 6 weeks after the start of alcohol administration. DNA synthesis was inhibited 24 hr after partial hepatectomy in rats given alcohol, but treatment with alanine and glutamine partially prevented this inhibition. To identify the mechanism of this effect, polyamine metabolism was studied. Administration of alcohol or alanine plus glutamine had no effect on the activity of ornithine decarboxylase, a rate-limiting enzyme of polyamine metabolism. In the liver, of the three polyamines, only the spermine concentration changed significantly. It decreased during long-term administration of alcohol, and this decrease was prevented by treatment with alanine and glutamine. The level of N(1)-acetylspermidine, the acetylated product of spermidine, was increased by alcohol, and its elevation was significantly less when alanine and glutamine were given. Hepatic spermidine/spermine N(1)-acetyltransferase, the key enzyme of polyamine acetylation, was induced by long-term administration of alcohol, and this induction was suppressed by alanine plus glutamine. The results suggest that treatment with alanine and glutamine can help to prevent the inhibition of liver regeneration caused by alcohol by maintaining the spermine level and suppressing the acetylation of spermidine.     

Phospholipase D activity in L1210 cells: a model for oleate-activated phospholipase D in intact mammalian cells

The effect of 2-aminobicyclo[2.2.1]heptan-2-carboxylic acid (BCH), an L-leucine nonmetabolizable analogue and an allosteric activator of glutamate dehydrogenase, on glucose and glutamine synthesis was studied in rabbit renal tubules incubated with alanine, aspartate or proline in the presence of glycerol and octanoate, i.e. under conditions of efficient glucose formation. With alanine+glycerol+octanoate the addition of BCH resulted in a stimulation of alanine and glycerol consumption, accompanied by an increased glucose, lactate and glutamine synthesis. In contrast, when alanine was substituted by either aspartate or proline, BCH altered neither glucose formation nor glutamine and glutamate synthesis, while an accelerated glycerol utilization was accompanied by a small increase in lactate production. In view of the BCH-induced changes in intracellular metabolite levels the acceleration of gluconeogenesis by BCH in the presence of alanine+glycerol+octanoate is probably due to (i) increased uptake of alanine via alanine aminotransferase, (ii) stimulation of phosphoenolpyruvate carboxykinase, a key-enzyme of gluconeogenesis, (iii) rise of glucose-6-phosphatase activity, as well as (iv) activation of the malate-aspartate shuttle resulting in an augmented glycerol utilization for lactate and glucose synthesis.     

Artificial salivas for in vitro studies of dental materials

In a preliminary study, levels of activity of enzymes involved in anaerobic glycerol catabolism by Enterobacter agglomerans grown in batch cultures regulated in a pH range of 6.5-8.0 were monitored. That study showed that activities of key enzymes of the downstream metabolism of glycerol--glyceradehyde-3-phosphate dehydrogenase (GAP-DH), lactate dehydrogenase and pyruvate formate lyase--were strongly dependent on the culture pH. To investigate the influence of pH on the physiology of the strain, E. agglomerans was grown anaerobically in a continuous culture supplied with glycerol as the sole carbon source and regulated at pH 8. A complete biochemical analysis was performed and was compared with that previously described for the continuous culture regulated at pH 7. A limitation of the glycolytic flux at the level of GAP-DH was demonstrated at high dilution rate, resulting in an overflow metabolism through the 1,3-propanediol formation pathway. Increasing the specific rate of glycerol consumption also resulted in enhanced lactate production due to limitation by the pyruvate decarboxylation step. Finally, changing the culture pH significantly modified the enzymatic profile of E. agglomerans, and it enabled the stability of the culture to be increased by preventing the accumulation in the fermentation broth of 3-hydroxypropionaldehyde, an inhibitory metabolite, when the glycerol supply was suddenly increased.     

Studies on the formation of alpha-amylase by Thermomonospora vulgaris

Conditions affecting the formation of alpha-amylase by static cultures of the thermophilic actinomycete Thermomonospora vulgaris were studied. The organism failed to grow under submerged culture conditions or when the culture medium was devoid of CaCO3-alpha-Amylase was produced during the logarithmic phase of growth and maximum yield was obtained after 3 to 9 days of incubation. Growth and amylase formation took place only in a range from 45 degrees to 55 degrees C; optimum temperature was 55 degrees C. Of the tested carbon sources only starch induced enzyme formation. Maximum enzyme yield was obtained when starch concentration of the medium was 2% and when ammonium citrate served as a nitrogen source. Crushed clay pots could substitute for CaCO3 of the medium, but growth and amylase yield were less.     

Influence of iron-limited continuous culture on physiology and virulence of Legionella pneumophila

A virulent strain of Legionella pneumophila serogroup 1, subgroup Pontiac, was grown in continuous culture at a constant growth rate under iron-replete and iron-limited conditions. Iron limitation was achieved by the removal of ferrous sulfate and hemin from the chemically defined medium. Residual contaminating iron, 0.45 microM, was sufficient to support iron-limited growth. Typical iron-replete cultures metabolized 3.3 microM iron. Serine provided the principal source of carbon and energy for both cultures, although iron-replete cultures also depleted a number of other amino acids. There was a 40% decrease in culture biomass under iron-restricted conditions. Iron limitation did not significantly affect carbohydrate metabolism, with the molar growth yield for carbon (Ycarbon) comparable for both cultures. However, under iron-limited conditions a sixfold increase in Yiron correlated with a significant decrease in the iron content of the biomass, as the culture utilized the available iron more efficiently. Highly pleomorphic iron-replete cultures became uniform cultures of short fine rods when adapted to iron-deficient conditions. In addition to the morphological and physiological changes, iron limitation had a critical effect on culture virulence. The virulence of this strain was significantly (P < 0.05) reduced when the culture was subjected to iron-limited conditions. This phenomenon was reversible, with a significant increase in culture virulence upon reversion to iron-replete conditions. When compared in an in vitro macrophage assay, the number of culturable avirulent iron-limited cells located intracellularly after infection was significantly lower than for the virulent replete and control cultures. These results further support the role of environmental parameters in regulating the virulence of L. pneumophila.     

Diverting Electron Fluxes to Hydrogen in Mixed Anaerobic Communities Fed with Glucose and Unsaturated C18 Long Chain Fatty Acids

Hydrogen (H2) production was maximized and methane (CH4) formation was minimized in a mixed anaerobic culture which was maintained at 21°C and fed glucose plus unsaturated long chain fatty acids (LCFAs). The initial pH in the batch reactors was 7.8±0.2. The two LCFAs under consideration included linoleic acid (LA) (two C=C bonds) and oleic acid (OA) (one C=C bond). Hydrogen production was observed when glucose was injected on Day 0 and again after Day 4. The H2 yield in cultures fed LA was less than those receiving OA. The H2 yield reached a maximum of approximately 1.1?mol?H2?mol?1 glucose when the LA level was 2,000?mg?L?1. In the case of OA, a maximum yield of 1.3?mol?H2?mol?1 glucose was attained with 2,000?mg?L?1. The inhibition caused by the addition of LA or OA diverted a fraction of electrons toward proton reduction. Under maximum H2 production conditions in the LA fed cultures the acetate production pathway was repressed, while in cultures fed OA the acetate pathway was dominant. The amount of CH4 produced decreased with increasing H2 production and the major volatile fatty acids detected were acetate, propionate and butyrate. Small quantities of formate were detected only in cultures fed LA after the first glucose injection. As the LCFA concentration increased, the initial glucose degradation rate decreased.     

High-density hybridoma perfusion culture. Limitation vs inhibition

Because our earlier work indicated a strong correlation between specific antibody productivity and cell density in perfusion culture, we conducted experiments to determine the optimum means of increasing cell density while maintaining high antibody productivity. The rates of medium supply and waste removal were varied to determine whether cell density was limited or inhibited, and whether a diffusable substance could be responsible for the correlation between antibody productivity and cell density. Nutrient supply was found to be a stronger determinant of cell density than waste removal; however, the rate of waste removal had a greater effect on cell growth at lower cell densities. Even at noninhibitory levels of ammonia and lactate, cellular metabolism was regulated to minimize their concentrations at lowered rates of waste removal. Separate step changes in glucose and glutamine resulted in increased cell density and antibody concentration. Specific antibody productivity increased following the step in glutamine, but not glucose. Both steps caused changes in cellular metabolism that prevented the levels of lactate and ammonia from reaching toxic levels.     

In vivo investigation of glutamate-glutamine metabolism in hyperammonemic monkey brain using 13C-magnetic resonance spectroscopy

To investigate the metabolism of glutamate and glutamine in living monkey brain, a system of in vivo 13C magnetic resonance spectroscopy (MRS) using 1H-decoupled 13C spectroscopy combined with monitoring temperature changes in the brain by MR phase mapping was developed. Serial 13C-NMR spectra of the amino acids glutamate and glutamine were acquired non-invasively over 4 h from anesthetized monkey brain after the intravenous administration of [1-13C]glucose (0.5-1.0 g/kg). In the acute hyperammonemic state induced by the administration of ammonium acetate (77 mg/kg bolus), it was observed that 13C incorporation into glutamine-4 was clearly accelerated, without changes of 13C incorporation into glutamate-4. During hyperammonemia, it was shown directly by [2-13C]glucose administration that the anaplerotic pathway for the TCA cycle was also augmented, contributing to the formation of glutamine in the astroglia.     

Characteristics and efficiency of glutamine production by coupling of a bacterial glutamine synthetase reaction with the alcoholic fermentation system of baker's yeast

Glutamine production with bacterial glutamine synthetase (GS) and the sugar-fermenting system of baker's yeast for ATP regeneration was investigated by determining the product yield obtained with the energy source for ATP regeneration (i.e., glucose) for yeast fermentation. Fructose 1,6-bisphosphate was accumulated temporarily prior to the formation of glutamine in mixtures which consisted of dried yeast cells, GS, their substrate (glucose and glutamate and ammonia), inorganic phosphate, and cofactors. By an increase in the amounts of GS and inorganic phosphate, the amounts of glutamine formed increased to 19 to 54 g/liter, with a yield increase of 69 to 72% based on the energy source (glucose) for ATP regeneration. The analyses of sugar fermentation of the yeast in the glutamine-producing mixtures suggested that the apparent hydrolysis of ATP by a futile cycle(s) at the early stage of glycolysis in the yeast cells reduces the efficiency of ATP utilization. Inorganic phosphate inhibits phosphatase(s) and thus improves glutamine yield. However, the analyses of GS activity in the glutamine-producing mixtures suggested that the higher concentration of inorganic phosphate as well as the limited amount of ATP-ADP caused the low reactivity of GS in the glutamine-producing mixtures. A result suggestive of improved glutamine yield under the conditions with lower concentrations of inorganic phosphate was obtained by using a yeast mutant strain that had low assimilating ability for glycerol and ethanol. In the mutant, the activity of the enzymes involved in gluconeogenesis, especially fructose 1,6-bisphosphatase, was lower than that in the wild-type strain.