Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions.

Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon- and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-1, was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 h-1 obtained under single glucose limitation.     

The metabolic network of Lactococcus lactis: distribution of (14)C-labeled substrates between catabolic and anabolic pathways

Lactococcus lactis NCDO 2118 was grown in a simple synthetic medium containing only six essential amino acids and glucose as carbon substrates to determine qualitatively and quantitatively the carbon fluxes into the metabolic network. The specific rates of substrate consumption, product formation, and biomass synthesis, calculated during the exponential growth phase, represented the carbon fluxes within the catabolic and anabolic pathways. The macromolecular composition of the biomass was measured to distribute the global anabolic flux into the specific anabolic pathways. Finally, the distribution of radiolabeled substrates, both into the excreted fermentation end products and into the different macromolecular fractions of biomass, was monitored. The classical end products of lactic acid metabolism (lactate, formate, and acetate) were labeled with glucose, which did not label other excreted products, and to a lesser extent with serine, which was deaminated to pyruvate and represented approximately 10% of the pyruvate flux. Other minor products, keto and hydroxy acids, were produced from glutamate and branched-chain amino acids via deamination and subsequent decarboxylation and/or reduction. Glucose labeled all biomass fractions and accounted for 66% of the cellular carbon, although this represented only 5% of the consumed glucose.     

Fluxes and enzyme activities in central metabolism of myeloma cells grown in chemostat culture

Activities of enzymes in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and glutaminolysis have been determined in the mouse myeloma SP2/0.Ag14. Cells were grown on IMDM medium with 5% serum in steady-state chemostat culture at a fixed dilution rate of 0.03 h-1. Three culture conditions, which differed in supply of glucose and oxygen, were chosen so as to change catabolic fluxes in the central metabolism, while keeping anabolic fluxes constant. In the three steady-state situations, the ratio between specific rates of glucose and glutamine consumption differed by more than twentyfold. The specific rates of glucose consumption and lactate production were highest at low oxygen supply, whereas the specific rate of glutamine consumption was highest in the culture fed with low amounts of glucose. Under low oxygen conditions, the specific production of ammonia increased and the consumption pattern of amino acids showed large changes compared with the other two cultures. For the three steady states, activities of key enzymes in glycolysis, the pentose phosphate pathway, glutaminolysis, and the TCA cycle were measured. The differences in the in vivo fluxes were only partially reflected in changes in enzyme levels. The largest differences were observed in the levels of glycolytic enzymes, which were elevated under conditions of low oxygen supply. High activities of phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) in all cultures suggest an important role for this enzyme as a link between glutaminolysis and glycolysis. For all enzymes, in vitro activities were found that could accommodate the estimated maximum in vivo fluxes. These results show that the regulation of fluxes in central metabolism of mammalian cells occurs mainly through modulation of enzyme activity and, to a much lesser extent, by enzyme synthesis.     

Effects of dissolved oxygen on hybridoma cell growth, metabolism, and antibody production kinetics in continuous culture.

The effects of dissolved oxygen concentration (DO) on hybridoma cell physiology were examined in a continuous stirred tank bioreactor with a murine hybridoma cell line (167.4G5.3). Dissolved oxygen concentration was varied between 0% and 100% air saturation. Cell growth and viability, carbohydrate, amino acid, and energy metabolism, oxygen uptake, and antibody production rates were investigated. Cell growth was inhibited at both high and low DO. Cells could grow at 0% DO and maintain viability under a nitrogen atmosphere. Cell viability was higher at low DO. Glucose, glutamine, and oxygen consumption rates changed little at DO above 1% air saturation. However, the metabolic uptake rates changed below 1% DO, where growth became oxygen limited, and a Km value of 0.6% DO was obtained for the specific oxygen uptake rate. The metabolic rates of glucose, glutamine, lactate, and ammonia increased 2-3-fold as the DO dropped from 1% to 0%. Amino acid metabolism followed the same general pattern as that of glutamine and glucose. Alanine was the only amino acid produced. The consumption rates of amino acids changed little above 1% DO, but under anaerobic conditions the consumption rates of all amino acids increased severalfold. Cells obtained most of their metabolic energy from glutamine oxidation except under oxygen limitation, when glucose provided most of the energy. The calculated ATP production rate was only slightly influenced by DO and rose at 0% DO. Antibody concentration was highest at 35% DO, while the specific antibody production rate was insensitive to DO.     

Calorimetric and stoichiometric analysis of growth of Kluyveromices fragilis in continuous culture: nitrogen limitation imposed upon carbon-limited growth

The calorimetric response of the yeast Kluyveromices fragilis was investigated for growth in continuous culture where nitrogen limitation was imposed on a carbon-limited culture. Calorimetric measurements were combined with off gas analysis, measurements of biomass, substrate and product concentrations, elemental biomass composition, and heat production to study the physiological response of K. fragilis. Regions where both carbon and nitrogen limited growth, were found over a broad range of dilution rates and feed carbon-to-nitrogen ratios. The principle mechanism by which K. fragilis accommodated regions of dual carbon and nitrogen limitation was by partial decoupling of the anabolic and catabolic pathways. When the culture was only nitrogen-limited, increased decoupling of the two pathways was observed. The principal effect of the decoupling was an increased catabolic consumption of glucose, generating an increased heat yield. The preferred way to process the excess glucose was through respiration but the cells were also capable of fermenting a small percentage of the excess glucose in specific cases where the dissolved oxygen partial pressure approached zero. In addition, these results were qualitatively compared to similar studies on Saccharomices cerevisiae. The two yeasts were similar in their ability to accommodate dual limitation by uncoupling anabolic biomass formation from substrate consumption. The two yeasts were dissimilar in how the catabolic substrate was processed. For S. cerevisiae the presence of a bottleneck in the respiration pathway dictated that the majority of the catabolic glucose consumption was by fermentation. For K. fragilis, the lack of a bottleneck in the respiration pathway dictated that the majority of catabolic glucose substrate consumption was by respiration.     

Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxygen-limited conditions in chemostat cultures.

Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolic and energetic control of Pseudomonas mendocina growth during transitions from aerobic to oxygen-limited conditions in chemostat cultures.

Several metabolic fluxes were analyzed during gradual transitions from aerobic to oxygen-limited conditions in chemostat cultures of Pseudomonas mendocina growing in synthetic medium at a dilution rate of 0.25 h-1. P. mendocina growth was glucose limited at high oxygen partial pressures (70 and 20% pO2) and exhibited an oxidative type of metabolism characterized by respiratory quotient (RQ) values of 1.0. A similar RQ value was obtained at low pO2 (2%), and detectable levels of acetic, formic, and lactic acids were determined in the extracellular medium. RQs of 0.9 +/- 0.12 were found at 70% pO2 for growth rates ranging from 0.025 to 0.5 h-1. At high pO2, the control coefficients of oxygen on catabolic fluxes were 0.19 and 0.22 for O2 uptake and CO2 production, respectively. At low pO2 (2%), the catabolic and anabolic fluxes were highly controlled by oxygen. P. mendocina showed a mixed-type fermentative metabolism when nitrogen was flushed into chemostat cultures. Ethanol and acetic, lactic, and formic acids were excreted and represented 7.5% of the total carbon recovered. Approximately 50% of the carbon was found as uronic acids in the extracellular medium. Physiological studies were performed under microaerophilic conditions (nitrogen flushing) in continuous cultures for a wide range of growth rates (0.03 to 0.5 h-1). A cell population, able to exhibit a near-maximum theoretical yield of ATP (YmaxATP = 25 g/mol) with a number of ATP molecules formed during the transfer of an electron towards oxygen along the respiration chain (P/O ratio) of 3, appears to have adapted to microaerophilic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

An anabolic state in the heart induced by arginine intubation

Using rat heart perfusion, we found that an anabolic state can be induced with a medium which includes glucose, carnitine, branched-chain amino acids and arginine after arginine intubation at a dose of 250 mg/kg body weight. It showed diminished levels of glutamine, glutamate, branched-chain oxoacids and phenylalanine (a marker of heart protein metabolism) release, reflecting anabolic changes occurring in the myocardium. While ornithine intubation caused a catabolic state in which the release of alanine and glutamate was increased but phenylalanine release was unchanged. This anabolic state may be a useful model providing for myocardial protection.     

Metabolism and Energetics of Lactococcus lactis during Growth in Complex or Synthetic Media

When Lactococcus lactis was grown in various complex or synthetic media, the fermentation of glucose remained homolactic whatever the medium used, with a global carbon balance of about 87%. Moreover, the nitrogen balance was not equilibrated, indicating that some amino acids led to the production of unknown nitrogen-containing carbon compounds while part of the glucose might contribute to anabolic pathways. In minimal medium containing six amino acids, a high concentration of serine was deaminated to pyruvate. This did not occur in more complete media, suggesting the presence of a regulation of this phenomenon by an amino acid. Ammonia produced during serine consumption was partly reconsumed after serine exhaustion. The values for biomass yield and biomass yield relative to ATP (Y(infATP)), the maximal growth rate, the specific rate of glucose consumption, and the corresponding rate of ATP synthesis all increased with the complexity of the medium, amino acid composition having the most pronounced effect. The Y(infATP) values were shown to range from 6.6 to 17.6 g of biomass(middot)mol of ATP(sup-1) on minimal and complex media.     

The transient responses of hybridoma cells to nutrient additions in continuous culture: II. Glutamine pulse and step changes

The transient and steady-state responses of hybridoma growth and metabolism to glutamine pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The specific glutamine consumption rate increased rapidly after all glutamine additions, but the responses of the glucose and oxygen consumption rates and the cell concentration were found to depend on the intial feed glutamine concentration. The glucose consumption rate was 1.4-10.9 times that of glutamine, and serine and branched-chain amino acids were consumed in larger amounts at the higher glucose: glutamine uptake ratios. It was estimated that maintenance accounted for ca. 60% of the cellular ATP requirements at specific growth rates ranging from 0.57 to 0.68 day(-1).     

The regulatory effects of growth rate and cyclic AMP levels on carbon catabolism and respiration in Escherichia coli K-12.

Cyclic AMP levels in glucose and succinate-limited and ammonia-limited glucose-containing continuous cultures of Escherichia coli were measured at different bacterial growth rates. Intracellular cyclic AMP concentrations were fairly constant (about 5 micrometer) at all dilution rates used when glucose was limiting. In ammonia-limited glucose cultures the cyclic AMP content was much lower (about 0.3 micrometer). In succinate-limited cultures cyclic AMP levels fell from 2.7 to 0.8 micrometer as dilution rate increased from 0.05 to 0.4 h-1. The effects of cyclic AMP on respiratory and carbon catabolic enzyme levels were studied. There was no indication of a direct cyclic AMP involvement in the regulation of these cellular functions. It seems more likely that the variations in enzyme levels observed resulted from variation of the specific growth rate of cultures.     

Kinetics of substrate utilization in adenine-limited chemostat cultures of Bacillus subtilis KYA741.

The specific rates of limiting substrate utilization were investigated in adenine- or glucose-limited chemostat cultures of Bacillus subtilis KYA741, an adenine-requiring strain, at 37 degrees C. With the glucose-limited cultures, the specific rate of glucose consumption versus dilution rate gave a linear relationship from which the true growth yield and maintenance coefficient were determined to be 0.09 mg of bacteria per mg of glucose and 0.2 mg of glucose per mg of bacteria per h, respectively. With the adenine-limited cultures, adenine as the limiting substrate was not completely consumed at lower dilution rates (e.g., D less than 0.1), unlike in the glucose-limited cultures. When a linear relationship of specific rate of adenine consumption versus dilution rate was extrapolated to zero dilution rate, a negative value for the specific rate of adenine consumption, -0.01 mg of adenine per mg of bacteria per h, was obtained, giving a true growth yield for adenine of 5.2 mg of bacteria per mg of adenine. On the other hand, the maintenance coefficient of oxygen uptake gave a positive value of 8.1 x 10(-3) mmol/mg of bacteria per h. Based on previous results showing that adenine is resupplied by lysing cells, we developed kinetic models of adenine utilization and cell growth that gave a good estimation of the peculiar behavior of cell growth and adenine utilization in adenine-limited chemostat cultures.     

A re-assessment of bacterial growth efficiency: the heat production and membrane potential of Streptococcus bovis in batch and continuous culture

Glucose-limited, continuous cultures (dilution rate 0.1 h^-1) of Streptococcus bovis JB1 fermented glucose at a rate of 3.9 mol mg protein^-1 h^-1 and produced acctate, formate and ethanol. Based on a maximum ATP yield of 32 cells/mol ATP (Stouthamer 1973) and 3 ATP/glucose, the theoretical glucose consumption for growth would have been 2.1 mol mg protein^-1 h^-1. Because the maintenance energy requirement was 1.7 mol/mg protein/h (Russell and Baldwin 1979), virtually all of the glucose consumption could be explained by growth and maintenance and the Y_ATP was 30. Glucose-limited, continuous cultures produced heat at a rate of 0.29 mW/mg protein, and this value was similar to the enthalpy change of the fermentation (0.32 mW/mg protein). Batch cultures (specific growth rate 2.0 h^-1) fermented glucose at a rate of 81 mol mg protein^-1 h^-1, and produced only lactate. The heat production was in close agreement with the theoretical enthalpy change (1.72 versus 1.70 mW/mg protein), but only 80% of the glucose consumption could be accounted by growth and maintenance. The Y_ATP of the batch cultures was 25. Nitrogen-limited, glucose-excess, non-growing cultures fermented glucose at a rate of 6.9 mol mg protein^-1 h^-1, and virtually all of the enthalpy for this homolactic fermentation could be accounted as heat (0.17 mW/mg protein). The nitrogenlimited cultures had a membrane potential of 150 mV, and nearly all of the heat production could be explained by a futile cycle of protons through the cell membrane (watts = amperes x voltage where H⁺/ATP was 3). The membrane voltage of the nitrogen-limited cells was higher than the glucose-limited continuous cultures (150 versus 80 mV), and this difference in voltage explained why nitrogen-limited cultures consumed glucose faster than the maintenance rate. Batch cultures had a membrane potential of 100 mV, and this voltage could not account for increased glucose consumption (more than growth plus maintenance). It appears that another mechanism causes the increased heat production and lower growth efficiency of batch cultures.     

Quantitative comparison of lactose and glucose utilization in Bifidobacterium longum cultures

In batch cultures, Bifidobacterium longum SH2 has a higher final cell concentration and greater substrate consumption when grown on lactose versus glucose. Continuous cultures were used to compare lactose and glucose utilization by B. longum quantitatively. In the continuous culture, the estimated maintenance coefficients (m) were similar when on lactose and glucose; the maximum cell yield coefficient (Y(X/S)(max)) was higher on lactose; and the specific consumption rate of lactose (q(S)) was lower than that of glucose. Assuming that cell growth followed the Monod model, the maximum specific growth rates (mu(max)) and saturation constants (K(S)) in lactose and glucose media were determined using the Hanes-Woolf plots. The respective values were 0.40 h(-)(1) and 78 mg/L for lactose and 0.46 h(-)(1) and 697 mg/L for glucose. The kinetic parameters of the continuous cultures showed that B. longum preferred lactose to glucose, although the specific consumption rate of glucose was higher than that of lactose.     

Amino acid regulation of insulin action in isolated adipocytes. Selective ability of amino acids to enhance both insulin sensitivity and maximal insulin responsiveness of the protein synthesis system

Using the number and concentration of amino acids in Dulbecco's modified Eagle's medium as reference (DMEM = 100%), we found that a maximally effective concentration of insulin (10 ng/ml) stimulated protein synthesis by 125% over basal rate in the presence of 50% amino acids (EC50 = 19%), but by only 48% in amino acid-free buffer. Moreover, time course experiments revealed that amino acid regulation of insulin action was very rapid (t1/2 of 9.5 min) and readily reversible (less than 30 min). This effect was specific in that basal rates of protein synthesis were unaltered by amino acids. A second effect of amino acids was to markedly enhance insulin sensitivity of the protein synthesis system in a dose-dependent manner. Thus, the half-maximally effective concentrations of insulin required to stimulate protein synthesis fell from 0.43 to 0.25 to 0.15 ng/ml in the presence of 0, 50, and 150% amino acids. Neither insulin sensitivity nor maximal insulin responsiveness of the glucose transport system was altered by amino acids, nor did amino acids affect the insulin binding capacity of cells. When we divided the 14 amino acids found in DMEM into two groups, we found that one group of 7 amino acids had little or no effect on insulin sensitivity or responsiveness, whereas the other group was fully active (a 157% increase in insulin responsiveness, ED50 of 0.21 ng/ml versus a 68% increase, ED50 of 0.51 ng/ml, with no amino acids). Isoleucine and serine together increased both insulin sensitivity and responsiveness to 60-70% of that seen with the full complement of amino acids. In conclusion: 1) amino acids modulate insulin action by enhancing maximal insulin responsiveness and insulin sensitivity of the protein synthesis system, and the regulatory site of amino acid action appears to be distal to the common signal pathway, within the insulin action-protein synthesis cascade, and 2) the effects of amino acids are specific, in that basal rates of protein synthesis are unaffected, only certain amino acids influence insulin action, and amino acids fail to alter insulin binding or the insulin-responsive glucose transport system. These studies, together with those in the companion paper, demonstrate that the pleiotropic actions of insulin on enhancing glucose uptake and protein synthesis are mediated through divergent pathways that can be independently regulated.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Metabolic flux analysis for optimizing the specific growth rate of recombinant <Emphasis Type="Italic">Aspergillus niger</Emphasis>

A comprehensive metabolic network comprising three intracellular compartments (cytoplasm, mitochondrion and peroxisome) was developed for Aspergillus niger. The metabolic flux network includes carbohydrate and amino acid metabolism in both anabolic and catabolic reactions. Linear programming was used for the optimization of the specific growth rates in combination with 37 measured input and output fluxes of the key metabolites to evaluate corresponding intracellular flux distributions throughout the batch fermentations. Logarithmic sensitivity analysis revealed that the addition of proline, alanine and glutamate benefited growth in defined media. The experimental observations and flux analysis showed that tyrosine was a potential candidate for biomass production improvement. Model predictions was verified by conducting batch and fed-batch fermentations and it was found that the addition of the four amino acids according to the predetermined schedule resulted in a 44 and 41% improvements in biomass and recombinant protein productions, respectively.     

Control of mixed-substrate utilization in continuous cultures of Escherichia coli

The chemostat culture technique was used to study the control mechanisms which operate during utilization of mixtures of glucose and lactose and glucose and l-aspartic acid by populations of Escherichia coli B6. Constitutive mutants were rapidly selected during continuous culture on a mixture of glucose and lactose, and the beta-galactosidase level of the culture increased greatly. After mutant selection, the specific beta-galactosidase level of the culture was a decreasing function of growth rate. In cultures of both the inducible wild type and the constitutive mutant, glucose and lactose were simultaneously utilized at moderate growth rates, whereas only glucose was used in the inducible cultures at high growth rates. Catabolite repression was shown to be the primary mechanism of control of beta-galactosidase level and lactose utilization in continuous culture on mixed substrates. In batch culture, as in the chemostat, catabolite repression acting by itself on the lac enzymes was insufficient to prevent lactose utilization or cause diauxie. Interference with induction of the lac operon, as well as catabolite repression, was necessary to produce diauxic growth. Continuous cultures fed mixtures of glucose and l-aspartic acid utilized both substrates at moderate growth rates, even though the catabolic enzyme aspartase was linearly repressed with increasing growth rate. Although the repression of aspartase paralleled the catabolite repression of beta-galactosidase, l-aspartic acid could be utilized even at very low levels of the catabolic enzyme because of direct anabolic incorporation into protein.     

Maintaining rapid growth in moderate-density Escherichia coli fermentations

A novel feeding strategy that prolongs rapid growth rates for Escherichia coli fermentations to moderately high cell density is presented. High-density fermentations are a common and successful means of producing biological products. However, acetate accumulation can be a substantial problem in these procedures. To avoid this problem, many feeding strategies and host modifications have been developed, but all result in relatively low growth rates. If a faster growth rate could be maintained, the growth phase of the process would be shortened, leading to increased productivity. It is also possible that the subsequent specific production rate could be enhanced by growing the early culture at a faster rate. We have developed a procedure to enable rapid growth to a cell density of 20 g/L and have used cell-free protein synthesis to evaluate the relative potential of the resulting cells for producing recombinant proteins. The method uses glucose pulses and the duration of the dissolved oxygen response to calculate the appropriate glucose feed rate based on the glucose demand of the culture. Amino acids and vitamins were supplied in the medium to increase the growth rate. We were able to sustain a growth rate of 0.8/h up to 20 g/L dry cell weight without significant acetate accumulation. Analysis of amino acid consumption indicates that cell composition is an accurate predictor of amino acid demand for most amino acids. Cell-free protein synthesis was used to compare the protein production potential of the high-density cultures with that of cells grown in complex medium and harvested at low cell density and maximum growth rate. Protein production for the extract from the controlled, high-density fermentations was 950 mg/L compared with 860 mg/L for the low-density control. Therefore, the new control procedure has promising potential for developing rapid and productive industrial fermentations.