Effect of different carbon sources on the production of succinic acid using metabolically engineered Escherichia coli

Succinic acid (SA) is an important platform molecule in the synthesis of a number of commodity and specialty chemicals. In the present work, dual-phase batch fermentations with the E. coli strain AFP184 were performed using a medium suited for large-scale industrial production of SA. The ability of the strain to ferment different sugars was investigated. The sugars studied were sucrose, glucose, fructose, xylose, and equal mixtures of glucose and fructose and glucose and xylose at a total initial sugar concentration of 100 g L-1. AFP184 was able to utilize all sugars and sugar combinations except sucrose for biomass generation and succinate production. For sucrose as a substrate no succinic acid was produced and none of the sucrose was metabolized. The succinic acid yield from glucose (0.83 g succinic acid per gram glucose consumed anaerobically) was higher than the yield from fructose (0.66 g g-1). When using xylose as a carbon source, a yield of 0.50 g g-1 was obtained. In the mixed-sugar fermentations no catabolite repression was detected. Mixtures of glucose and xylose resulted in higher yields (0.60 g g-1) than use of xylose alone. Fermenting glucose mixed with fructose gave a lower yield (0.58 g g-1) than fructose used as the sole carbon source. The reason is an increased pyruvate production. The pyruvate concentration decreased later in the fermentation. Final succinic acid concentrations were in the range of 25-40 g L-1. Acetic and pyruvic acid were the only other products detected and accumulated to concentrations of 2.7-6.7 and 0-2.7 g L-1. Production of succinic acid decreased when organic acid concentrations reached approximately 30 g L-1. This study demonstrates that E. coli strain AFP184 is able to produce succinic acid in a low cost medium from a variety of sugars with only small amounts of byproducts formed.     

Growth characteristics of Escherichia coli HB101[pGEc47] on defined medium

This paper shows that differences in growth behavior of Escherichia coli strain HB101 and strain HB101[pGEc47] can be related to yeast extract-enriched medium rather than plasmid properties. An optimal medium for growth of E. coli HB101[pGEc47] was designed based on the individual yield coefficients for specific medium components (NH4+ 6 g g-1, PO43- 14 g g-1, SO42- 50 g g-1). The yield coefficient for L-leucine depends on the glucose content of the medium (20 g g-1 for 3% glucose, 40 g g-1 for 1% glucose) and the yield coefficient for L-proline depends on the cultivation mode (20 g g-1 for batch cultivation, 44 g g-1 for continuous cultivation). Growth on defined medium after medium optimization is as rapid as on complex medium (0. 42-0.45 h-1). The critical dilution rate (DR) in the defined medium above which undesired production of acetic acid occurs is in the range of 0.23-0.26 h-1.     

Intracellular pH-based controlled cultivation of yeast cells: II. cultivation methodology

Intracellular pH (pH(i)) was measured on-line in a bioreactor using a fluorescent pH(i) indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pH(i) (FB-pH(i)) were performed. In FB-pH(i) cultivations, automated glucose additions were made to the culture in response to culture pH(i). The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g(-1) glucose in fed-batch pH(i) cultivations with 100 ppm glucose additions (FB-pH(i)-100 cultivation) vs. 0.48 g g(-1) glucose in batch] and cell yield was higher (0.54 g g(-1) glucose in FB-pH(i)-100 cultivation vs. 0.3 g g(-1) glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pH(i) from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pH(i), performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pH(i) cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pH(i) cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. (c) 1993 John Wiley & Sons, Inc.     

Physiological and morphological study of encapsulated Saccharomyces cerevisiae

The impact of encapsulation on the anaerobic growth pattern of S. cerevisiae CBS 8066 in a defined synthetic medium over 20 consecutive batch cultivations was investigated. In this period, the ethanol yield increased from 0.43 to 0.46 g/g, while the biomass and glycerol yields decreased by 58 and 23%, respectively. The growth rate of the encapsulated cells in the first batch was 0.13 h⁻¹, but decreased gradually to 0.01 h⁻¹ within the 20 sequential batch cultivations. Total RNA content of these yeast cells decreased by 39% from 90.3 to 55 mg/g, while the total protein content decreased by 24% from 460 to 350 mg/g. On the other hand, the stored carbohydrates, that is, glycogen and trehalose content, increased by factors of 4.5 and 4 within 20 batch cultivations, respectively. Higher biomass concentrations inside capsules led to a lower glucose diffusion rate through the membrane, and volumetric mass transfer coefficient for glucose was drastically decreased from 6.28 to 1.24 (cm³/min) by continuing the experiments. Most of the encapsulated yeast existed in the form of single and non-budding cells after long-term application.     

Heterotrophic high cell-density fed-batch cultures of the phycocyanin-producing red alga Galdieria sulphuraria

Growth and phycocyanin production in batch and fed-batch cultures of the microalga Galdieria sulphuraria 074G, which was grown heterotrophically in darkness on glucose, fructose, sucrose, and sugar beet molasses, was investigated. In batch cultures, specific growth rates and yields of biomass dry weight on the pure sugars were 1.08-1.15 day-1 and 0.48-0.50 g g-1, respectively. They were slightly higher when molasses was the carbon source. Cellular phycocyanin contents during the exponential growth phase were 3-4 mg g-1 in dry weight. G. sulphuraria was able to tolerate concentrations of glucose and fructose of up to 166 g L-1 (0.9 M) and an ammonium sulfate concentration of 22 g L-1 (0.17 M) without negative effects on the specific growth rate. When the total concentration of dissolved substances in the growth medium exceeded 1-2 M, growth was completely inhibited. In carbon-limited fed-batch cultures, biomass dry weight concentrations of 80-120 g L-1 were obtained while phycocyanin accumulated to concentrations between 250 and 400 mg L-1. These results demonstrate that G. sulphuraria is well suited for growth in heterotrophic cultures at very high cell densities, and that such cultures produce significant amounts of phycocyanin. Furthermore, the productivity of phycocyanin in the heterotrophic fed-batch cultures of G. sulphuraria was higher than is attained in outdoor cultures of Spirulina platensis, where phycocyanin is presently obtained.     

Facilitated Transport of Glucose from Blood into Peripheral Nerve

D-Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood-nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D-[14C]glucose, L-[14C]glucose, and [14C]3-O-methyl-D-glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D-glucose. The permeability-surface area product (PA) for D-[14C]glucose and [14C]3-O-methyl-D-glucose decreased, whereas the PA for L-[14C]glucose remained constant, as the perfusate concentration of D-glucose was increased. In the presence of no added unlabelled D-glucose in the perfusate, the PA for L-[14C]glucose equaled one-fifth the PA for D-[14C]glucose. These results demonstrate self-saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D-monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half-saturation constant and maximal rate of transport for the saturable component of D-glucose influx equaled 23 +/- 11 mumol X ml-1 and 6.6 +/- 3.2 X 10(-3) mumol X s-1 X g-1, respectively. The constant of nonsaturable glucose influx equaled 0.5 +/- 0.1 X 10(-4) s-1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D-glucose influx into nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physiological and morphological study of encapsulated Saccharomyces cerevisiae

The impact of encapsulation on the anaerobic growth pattern of S. cerevisiae CBS 8066 in a defined synthetic medium over 20 consecutive batch cultivations was investigated. In this period, the ethanol yield increased from 0.43 to 0.46 g/g, while the biomass and glycerol yields decreased by 58 and 23%, respectively. The growth rate of the encapsulated cells in the first batch was 0.13 h⁻¹, but decreased gradually to 0.01 h⁻¹ within the 20 sequential batch cultivations. Total RNA content of these yeast cells decreased by 39% from 90.3 to 55 mg/g, while the total protein content decreased by 24% from 460 to 350 mg/g. On the other hand, the stored carbohydrates, that is, glycogen and trehalose content, increased by factors of 4.5 and 4 within 20 batch cultivations, respectively. Higher biomass concentrations inside capsules led to a lower glucose diffusion rate through the membrane, and volumetric mass transfer coefficient for glucose was drastically decreased from 6.28 to 1.24 (cm³/min) by continuing the experiments. Most of the encapsulated yeast existed in the form of single and non-budding cells after long-term application.     

Optimization and stability of glucoamylase production by recombinant strains of Aspergillus niger in chemostat culture

When grown on a medium containing 5 g maltodextrin L-1, Aspergillus niger transformant N402[pAB6-10]B1, which has an additional 20 copies of the glucoamylase (glaA) gene, produced 320 +/- 8 mg (mean +/- S.E.) glucoamylase (GAM) L-1 in batch culture and 373 +/- 9 mg GAM L-1 in maltodextrin-limited chemostat culture at a dilution rate of 0.13 h-1. These values correspond to specific production rates (qp) of 5.6 and 16.0 mg GAM [g biomass]-1 h-1, respectively. In maltodextrin-limited chemostat cultures grown at dilution rates from 0.06 to 0.14 h-1, GAM was produced by B1 in a growth-correlated manner, demonstrating that a continuous flow culture system operated at a high dilution rate is an efficient way of producing this enzyme. In chemostat cultures grown at high dilution rates, GAM production in chemostat cultures was repressed when the limiting nutrient was fructose or xylose, but derepressed when the limiting nutrient was glucose (qp, 12.0), potassium (6.2), ammonium (4.1), phosphate (2.0), magnesium (1.5) or sulphate (0.9). For chemostat cultures grown at a dilution rate of 0.13 h-1, the addition of 5 g mycopeptone L-1 to a glucose-mineral salts medium resulted in a 64% increase in GAM concentration (from 303 +/- 12 to 496 +/- 10 mg GAM L-1) and a 37% increase in specific production rate (from 12.0 +/- 0.4 to 16.4 +/- 1.6 mg GAM [g biomass]-1 h-1). However, although recombinant protein production was stable for at least 948 h (191 generations) when A. niger B1 was grown in chemostat culture on glucose-mineral salts medium, it was stable for less than 136 h (27 generations) on medium containing mycopeptone. The predominant morphological mutants occurring after prolonged chemostat culture were shown to have selective advantage in the chemostat over the parental strain. Compared to their parental strains, two morphological mutants had similar GAM production levels, while a third had a reduced production level. Growth tests and molecular analysis revealed that the number of glaA gene copies in this latter strain (B1-M) was reduced, which could explain its reduced GAM production. Shake-flask cultures carried out with the various morphological mutants revealed that in batch culture all three strains produced considerably less GAM than their parent strains and even less than N402. We show that physiological changes in these morphological mutants contribute to this decreased level of GAM production.     

Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source

Growth, substrate consumption, metabolite formation, biomass composition and respiratory parameters of Kluyveromyces marxianus ATCC 26548 were determined during aerobic batch and chemostat cultivations, using mineral medium with glucose as the sole carbon source, at 30 degrees C and pH 5.0. Carbon balances closed within 95-101% in all experiments. A maximum specific growth rate of 0.56 h(-1), a biomass yield on glucose of 0.51 g g(-1), and a maximum specific consumption of oxygen of 11.1 mmol g(-1) h(-1) were obtained during batch cultures. The concentration of excreted metabolites was very low at the culture conditions applied, representing 6% of the consumed carbon at most. Acetate and pyruvate were excreted to a larger extent than ethanol under the batch conditions, and the protein content accounted for 54.6% of the biomass dry weight. Steady states were obtained during chemostats at dilution rates of 0.1, 0.25 and 0.5 h(-1). At the two former dilution rates, cells grew at carbon limitation and the biomass yield on glucose was similar to that obtained under the batch conditions. Metabolite formation was rather low, accounting for a total of 0.005 C-mol C-mol(-1) substrate. At 0.5 h(-1), although the biomass yield on glucose was similar to the value obtained under the above-mentioned conditions, the cultivation was not under carbon limitation. Under this condition, 2-oxoglutarate, acetate, pyruvate and ethanol were the prevalent metabolites excreted. Total metabolite formation only accounted to 0.056 C-mol C-mol(-1) of substrate. A very high protein and a low carbohydrate content (71.9% and 9.6% of biomass dry weight, respectively) were measured in cells under this condition. It is concluded that K. marxianus aligns with the so-called aerobic-respiring or Crabtree-negative yeasts. Furthermore, it has one of the highest growth rates among yeasts, and a high capacity of converting sugar into biomass, even when carbon is not the limiting nutrient. These results provide useful data regarding the future application of K. marxianus in processes aimed at the production of biomass-linked compounds, with high yields and productivities.     

Cell growth and enzyme synthesis of a mutant of Arthrobacter sp. (DSM 3747) used for the production of L-amino acids from D,L-5-monosubstituted hydantoins.

A microorganism with the ability to form L-tryptophan from D,L-5-(3-indolyl-methyl)hydantoin (D,L-5-IMH) was isolated and identified as Arthrobacter sp. (DSM 3747). After isolation of a mutant with high tryptophan production activity but low tryptophan degradation, cultural conditions were optimized to achieve high amounts of biomass with good specific activities concerning the enzymatic hydantoin-cleaving reactions. The ability of the microorganism to perform these bioconversions was found to be inducible by D,L-5-IMH as well as to be dependent on the presence of Mn2+. The highest specific D,L-5-IMH-cleaving activity of the cells was observed in the exponential phase of growth. The addition of yeast extract to the mineral salts medium was found to be essential for obtaining biomass concentrations of about 25 g l-1 cell dry mass by bioreactor cultivations. In order to obtain a constantly high growth rate, feeding of the C-source was pO2-controlled. The inducer D,L-5-IMH had to be continuously fed to prevent a decline of the L-tryptophan-forming enzyme activities, because it was subjected to degradation with the enzymes induced and higher concentrations of D,L-5-IMH aggravated the growth significantly. The synthesis of the enzymes was also inducible, when inducer and Mn2+ were not added until the late growth phase. Using this process, the consumption of D,L-5-IMH was reduced remarkably. So, under these conditions biomass concentrations of 25 g l-1 cell dry weight with a specific enzymatic activity of 0.20 mmol g-1 h-1 (tryptophan per dry mass per time) could be obtained within 13 h. Using 1 g l-1 of the chemically modified inducer D,L-5-(3-indolylmethyl)-3-N-methylhydantoin, which was not degradable by the microorganisms, a biomass concentration of 28 g l-1 cell dry weight with a specific activity of 0.34 mmol g-1 h-1 (tryptophan per dry mass per time) could be obtained within 28 h.     

The growth and nutrient utilization of the insect cell line Spodoptera frugiperda Sf9 in batch and continuous culture

The growth of the Spodoptera frugiperda cell line Sf9 was studied in batch and continuous culture. The results of batch cultivations showed that glucose was the preferred energy and carbon source limiting the cell density in both TNM-FH and IPL-41 media. Continuous culture using IPL-41-based feeding medium with different glucose (2.5, 5 and 10 g l⁻¹) and yeast extract concentrations (4, 8 and 16 g l⁻¹) showed that in serum-supplemented medium the maximum cell density was limited by glucose and yeast extract concentration. The transition to glucose limitation caused a decrease in growth rate and viability. A high cell density culture (18 × 10⁶ ml⁻¹) was obtained using a glucose concentration of 10 g l⁻¹ and a yeast extract concentration of 8 g l⁻¹ in the feeding medium. A yeast extract concentration of 16 g l⁻¹ inhibited growth. Unlike mammalian cell cultures, lactate, alanine and ammonia were not involved in growth inhibition. Lactate did not accumulate under aerobic conditions. Ammonia accumulation, if observed, was insignificant. The level of alanine synthesized and excreted into the culture medium never reached an inhibitory level. During glucose limitation alanine did not accumulate and ammonia was released. However, even in the presence of glucose significant amounts of Asp, Glu, Gln, Asn, Ser, Arg and Met were utilized for energy production. The amino groups of these amino acids were transferred to pyruvate or used for nucleic acid synthesis and excreted in the form of alanine into the culture medium. The consumption of His, Lys, Thr, Gly, Val, Leu, Phe, Tyr, Trp and Ile by growing Sf-9 cells was almost equal to their concentration in the biomass.     

Lysine and alanine transport in the perfused guinea-pig placenta

The characteristics of L-lysine transport were investigated at brush-border (maternal) and basal (fetal) sides of the syncytiotrophoblast in the term guinea-pig placenta artificially perfused either through the umbilical vessels in situ or through both circulations simultaneously. Cellular uptake, efflux and transplacental transfer were determined using a single-circulation paired-tracer dilution technique. Unidirectional L-[3H]lysine uptake (%) (perfusate lysine 50 microM) was high on maternal (M = 87 +/- 1) and fetal (F = 73 +/- 2) sides. L-[3H]Lysine efflux back into the ipsilateral circulation was asymmetrical (F/M ratio = 2.3) and transplacental flux occurred in favour of the fetal circulation. Unidirectional lysine influx kinetics (0.05-8.00 mM) gave Km values of 1.75 +/- 0.70 mM and 0.90 +/- 0.25 mM at maternal and fetal sides, respectively; corresponding Vmax values were 1.95 +/- 0.38 and 0.87 +/- 0.10 mumol.min-1.g-1. At both sides, lysine influx (50 microM) could be inhibited (about 60-80%) by 4 mM L-lysine and L-ornithine and less effectively (about 10-40%) by L-citrulline, L-arginine, D-lysine and L-histidine. At the basal side: (i) lysine influx kinetics were greatly modified in the presence of 10 mM L-alanine (Km = 6.25 +/- 3.27 mM; Vmax = 2.62 +/- 0.94 mumol.min-1.g-1), but unchanged by equimolar L-phenylalanine or L-tryptophan; (ii) in the converse experiments, lysine (10 mM) did not affect the kinetic characteristics for either L-alanine or L-phenylalanine; (iii) L-lysine and L-alanine influx kinetics were not dependent on the sodium gradient; (iv) the inhibition of L-[3H]lysine uptake by 4 mM L-homoserine was partially (60%) Na+-dependent. At the maternal side the kinetic characteristics for alanine influx were highly Na+-dependent, while lysine influx was partially Na+-dependent only at low concentrations (0.05-0.5 mM). Bilateral perfusion with 2,4-dinitrophenol (1 mM) reduced L-[3H]lysine uptake into the trophoblast and abolished transplacental transfer. It is suggested that lysine transport in the guinea-pig placenta is mediated by a specific transport system (y+) for cationic amino-acids. The asymmetry in the degree of sodium-dependency at both trophoblast membranes may in part explain the maternal-to-foetal polarity of placental amino-acid transfer in vivo.     

Determination of the maximum specific uptake capacities for glucose and oxygen in glucose-limited fed-batch cultivations of Escherichia coli

A simple pulse-based method for the determination of the maximum uptake capacities for glucose and oxygen in glucose limited cultivations of E. coli is presented. The method does not depend on the time-consuming analysis of glucose or acetate, and therefore can be used to control the feed rate in glucose limited cultivations, such as fed-batch processes. The application of this method in fed-batch processes of E. coli showed that the uptake capacity for neither glucose nor oxygen is a constant parameter, as often is assumed in fed-batch models. The glucose uptake capacity decreased significantly when the specific growth rate decreased below 0.15 h(-1) and fell to about 0.6 mmol g(-1) h(-1) (mmol per g cell dry weight and hour) at the end of fed-batch fermentations, where specific growth rate was approximately 0.02 h(-1). The oxygen uptake capacity started to decrease somewhat earlier when specific growth rate declined below 0.25 h(-1) and was 5 mmol g(-1) h(-1) at the end of the fermentations. The behavior of both uptake systems is integrated in a dynamic model which allows a better fitting of experimental values for glucose in fed-batch processes in comparison to generally used unstructured kinetic models.     

Effects of cycloheximide on protein degradation and gluconeogenesis in the perfused rat liver.

Cycloheximide at concentrations above 18 muM produced a 93% inhibition of total protein synthesis measured by valine incorporation in the perfused rat liver. Rates of protein degradation were estimated by perfusing livers prelabeled in vivo with L-[1-14C]valine with medium containing 15 mM L-valine. Thus labeled valine released from liver protein during perfusion was greatly diluted and reincorporation of label was minimized. Cycloheximide at 18 muM inhibited protein degradation by over 60%, after a delay of 15-20 min. Associated with these effects were dose-dependent increases in the rates of glucose and urea production. Glucose production increased 3 fold, from 0.54 +/- 0.07 in control to 1.85 +/- 0.24 mumol/min/100 g rat in cycloheximide-treated livers. Urea production increased from 0.24 +/- 0.02 to 0.62 +/- 0.06 mumol/min/100 g rat. No changes in liver glycogen or cyclic AMP content were seen. The data suggest that inhibition of protein synthesis provides an increased availability of intra-cellular amino acids and that many of these are rapidly degraded, yielding urea and glucose. This is supported by the fact that intracellular alanine levels were significantly increased following cycloheximide treatment. It is possible that the inhibition of protein degradation by cycloheximide is due to altered intra-cellular pools of amino acids or their metabolites.     

The effect of vanadate on glucose transport and metabolism in rat small intestine

The effect of sodium orthovanadate on the absorption, transmural transport and metabolism of glucose was studied by perfusion of isolated loops of rat jejunum in vitro. The presence of 1 mM vanadate in the serosal medium diminished absorption from 539 +/- 19 (n = 12) to 246 +/- 19 (P less than 0.001) mumol/h per g dry weight and transmural transport from 333 +/- 17 to 14 +/- 19 (P less than 0.001) mumol/h per g dry weight, whereas glucose utilisation was unaffected. The rate of release of lactate into the serosal medium was also diminished from 168 +/- 14 to 75 +/- 5 mumol/h per g dry weight (P less than 0.001). The observed rates were linear with respect to time and vanadate was effective within 5 min. In contrast, the rate of release of lactate into the luminal perfusate was strongly enhanced. Moreover, the progress curve showed a positive transient with an apparent lag time of 18.0 +/- 0.3 min, during which the rate increased to a value 9.2-times that of the control. Under the final steady-state conditions, the ratio of mucosal to serosal lactate production was 5.2 +/- 0.2 compared with 0.25 +/- 0.06 for the control, so that the effect of vanadate was to reverse the vectorial disposition of lactate. The concentration dependence of the effect of vanadate on absorption and metabolism was similar to that observed for the inhibition by vanadate of Na+/K+-ATPase activity in mucosal homogenates. The results are discussed in terms of the dissipation of transmembrane Na+ gradients as a result of the inhibition of the Na+/K+-ATPase.     

Calorimetric control of fed-batch cultures of Saccharomyces cerevisiae

Calorimetry has been used to control the glucose feeding in fed-batch cultures of S. cerevisiae in order to avoid ethanol formation and maintain a fully respiratory metabolism. Comparisons between batch and fed-batch cultivations showed that the former had a much lower growth yield. The growth yields for fed-batch cultivations were more than 30% higher than for batch cultures. However, energy balance calculations showed that a large part of the increase could be explained by the evaporation of ethanol during batch cultivations. When the growth yields obtained from the batch cultures were corrected for the evaporation of ethanol, the increase in growth yield for fed-batch cultures was about 10%.     

Muscle glucose uptake of obese Zucker rats trained at two different intensities

Exercise training reduces the muscle insulin resistance of the obese Zucker rat. The purpose of the present study was to determine whether the magnitude of this training response is exercise intensity specific. Obese Zucker rats were randomly divided into sedentary (SED), low-intensity (LI), and high-intensity (HI) exercise groups. For the LI rats, exercise training consisted of running on a rodent treadmill at 18 m/min up an 8% grade for 90 min. Rats in the HI group ran at 24 m/min up an 8% grade for four 17-min bouts with 3 min between bouts. Both exercise groups performed the same amount of work and trained 5 days/wk for 7 wk. To evaluate muscle insulin resistance, rat hindlimbs were perfused for 30 min with perfusate containing 6 mM glucose (0.15 mu Ci of D-[14C(U)] glucose/ml) and either a maximal (10.0 mU/ml) or a submaximal (0.50 mU/ml) insulin concentration. Perfusions were performed 48-56 h after the last exercise bout and a 12-h fast. In the presence of 0.5 mU/ml insulin, the rate of muscle glucose uptake was found to be significantly faster for the HI (9.56 +/- 0.66 mumol.h-1.g-1) than for the LI (7.72 +/- 0.65 mumol.h-1.g-1) and SED (6.64 +/- 0.44 mumol.h-1.g-1) rats. The difference in glucose uptake between the LI and SED rats was not significant. In the presence of 10.0 mU/ml insulin, the rate of glucose uptake was significantly faster for the HI (16.43 +/- 1.02 mumol.h-1.g-1) than for the LI rats (13.76 +/- 0.84 mumol.h-1.g-1) and significantly faster for the LI than for the SED rats (11.02 +/- 0.35 mumol.h-1.g-1).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of modified glucose uptake using genetic engineering techniques on high-level recombinant protein production in escherichia coli dense cultures

Reduction of acetate excretion using a modified cellular glucose uptake rate was examined. An Escherichia coli strain bearing a mutationin ptsG, a gene encoding enzyme II in glucose phosphotransferase system (PTS), was constructed and characterized. The growth rate of the mutant strain was slower than its parent in glucose defined medium, butwas not affected in complex medium. Experimental results using this mutant strain showed a significant improvement in culture performance in simple batch cultivations due to reduced acetate excretion through the modified glucose uptake. Both biomass and recombinant protein productivity were increased by more than 50% with the ptsG mutant when compared to the parent strain. Recombinant protein productivity by the newly constructed strain at a level of more than 1.6 g/L was attained consistently in a simple batch bioreactor. (c) 1994 John Wiley & Sons, Inc.     

Formation of poly-3-hydroxybutyrate by a soil microbial community during batch and heterocontinuous cultivation

Glucose added to soil as an extracellular source of carbon and energy was proved to be deposited into an intracellular polymer poly-3-hydroxybutyrate (PHB). Untreated soil samples contained PHB amounts corresponding to 1.56 –2.64 μg of crotonic acid per 1 g soil. During batch cultivation after addition of 1 % glucose the PHB content increased by 20-fold after 2 d and then decreased owing to the disappearance of glucose from the soil. Repeated additions of glucose did not bring about any significant increase in PHB content as compared with a single addition. In soil supplied continuously with 0.1 or 0.25 % glucose solution, the content of PHB increased, after an initial lag, gradually up to the 10th day. After 1-d cultivation the content of PHB in the batch system increased even in the presence of diammonium hydrogen phosphate. In a heterocontinuous system no PHB accumulation took place in the presence of this source of nitrogen and phosphorus as long as the C:N ratio of theadded substrate was 10: 1.