Regulation of sugar transport via the multiple sugar metabolism operon of Streptococcus mutans by the phosphoenolpyruvate phosphotransferase system.

In this report, we provide evidence that the transport of sugars in Streptococcus mutans via the multiple sugar metabolism system is regulated by the phosphoenolpyruvate phosphotransferase system. A ptsI-defective mutant (DC10), when grown on the multiple sugar metabolism system substrate raffinose, exhibited reduced growth, transport, and glycolytic activity with raffinose relative to the parent strain BM71. Inhibition of [3H]raffinose uptake was also observed in both BM71 and DC10 with increasing concentrations of glucose and the glucose analogs alpha-methyl glucoside and 2-deoxyglucose.     

Balanced nutrient fortification enables high-density hybridoma cell culture in batch culture

Cells of an in vitro culture system are not the same as for an in vivo system, metabolically and physiologically; ineffective utilization of nutrients occurs by cells in vitro. Therefore, a simpler approach is needed to examine closely and overcome differences between in vivo and in vitro cells.Recognizing the ineffectiveness of nutrient utilization in vitro, we have constructed, a balanced, fortified high-density medium based on RPMI 1640 medium previously optimized for relatively low-density cell culture. The high-density medium was used to cultivate a hybridoma line in a batch spinner flask culture. In this fortified medium, a hybridoma cell line 2c3.1 was cultivated to near 1 x 10(7) cells/mL in batch suspension culture. During the culture, glucose, glutamine, and 10 essential amino acids of concentrations five times richer than normal in the medium were almost thoroughly consumed. Combined analysis of these consumption profiles reveals that the balanced, fortified nutrient supply contributes much to cellular activity to overcome the limitations of in vitro cellular growth. Intermediate metabolites, such as ammonium ion and lactic acid, were produced over concentrations reported until now to be inhibitory. This observation suggests that the major conclusive factor against cellular growth over the critical cell density is not so-called inhibitory metabolites. As a result of the high-density culture, 5-8 times higher production of a monoclonal antibody for hepatitis B surface antigen (anti-HBs) was obtained.Active cellular consumption of all the essential nutrients and the corresponding production of MAb strongly support the potential of our approach to overcome the growth limitation of cells in vitro and to obtain high-density hybridoma cell culture.     

Regulation of sugar transport and metabolism in lactic acid bacteria

Abstract The phosphoenolpyruvate (PEP)-dependent lactose: phosphotransferase system (PTS), P-β-galactosidase, and enzymes of the d -tagatose-6P pathway, are prerequisite for rapid homolactic fermentation of lactose by Group N ('starter') streptococci. Moreover, the reactions of transport and catabolism constitute an open cycle in which ATP and lactic acid are metabolic products. The efficient and controlled operation of this cycle requires 'fine-control' mechanisms to ensure: (i) tight coupling between transport and energy-yielding reactions, (ii) co-metabolism of both glucose and galactose moieties of the disaccharide, and (iii) coordination of the rate of sugar transport to the rate of sugar catabolism. The elucidation of these fine-control mechanisms in intact cells of Streptococcus lactis has required the isolation of glucokinase (GK) and mannose-PTS defective mutants, the synthesis of novel lactose analogs, and the use of high resolution [³¹P]NMR spectroscopy. It has been established that PEP provides the crucial link between transport and energy-yielding reactions of the PTS: glycolysis cycle, and that both ATP-dependent glucokinase and PEP-dependent mannose-PTS can participate in the phosphorylation of intracellular glucose. Finally, evidence has been obtained in vivo, for modulation of pyruvate kinase activity in response to fluctuation in, concentrations of positive (FDP), and negative (P_i) effectors of the allosteric enzyme. Fine-control of pyruvate kinase activity may in turn regulate: (i) the distribution of PEP to either the PTS or ATP synthesis, (ii) overall activity of the PTS: glycolysis cycle, and (iii) the formation of the endogenous PEP-potential in starved organisms. The accumulation of non-metabolizable PTS sugars (e.g., 2-deoxy- d -glucose) by growing cells can perturb these fine-control mechanisms and, by establishment of a PEP-dissipating futile cycle, may result in bacteriostasis.     

Alterations in Taxol production in plant cell culture via manipulation of the phenylalanine ammonia lyase pathway

One approach to increasing secondary metabolite production in plant cell culture is to manipulate metabolic pathways to utilize more resources toward production of one desired compound or class of compounds, such as diverting carbon flux from competing secondary pathways. Since phenylalanine provides both the phenylisoserine side chain and the benzoyl moiety at C-2 of Taxol, we speculated that blockage of the phenylpropanoid pathway might divert phenylalanine into Taxol biosynthesis. We used specific enzyme inhibitors to target the first enzyme in the phenylpropanoid pathway, phenylalanine ammonia lyase (PAL), the critical control point for conversion of L-phenylalanine to trans-cinnamic acid. Cinnamic acid acted quickly in reducing PAL activity by 40-50%, without affecting total protein levels, but it generally inhibited the taxane pathway, reducing Taxol by 90% of control levels. Of the taxanes produced, 13-acetyl-9-dihydro-baccatin III and 9-dihydrobaccatin III doubled as a percentage of total taxanes in C93AD and CO93P cells treated with 0.20 and 0.25 mM cinnamic acid, when all other taxanes were lowered. The PAL inhibitor alpha-aminooxyacetic acid (AOA) almost entirely shut down Taxol production at both 0.5 and 1.5 mM, whereas L-alpha-aminooxy-beta-phenylpropionic acid (AOPP) had the opposite effect, slightly enhancing Taxol production at 1 microM but having no effect at 10 microM. The discrepancy in the effectiveness of AOA and AOPP and the lack of effect with addition of phenylalanine or benzoic acid derivatives further indicates that the impact of cinnamic acid on Taxol is related not to its effect on PAL but rather to a specific effect on the taxane pathway. On the basis of these results, a less direct route for inhibiting the phenylpropanoid pathway may be required to avoid unwanted side effects and potentially enhance Taxol production.     

Increased rates of sugar transport in Saccharomyces cerevisiae a result of sugar metabolism

Preincubation of yeast cells with glucose or other metabolic energy sources increased the rate of sorbose efflux 2- or 3-fold. Stimulated rates persisted for several h, decreasing slowly. They were approximately halved by including K_m concentrations of highly competitive sugars such as deoxyglucose, glucose, fructose and mannose in sorbose efflux suspensions, and were greatly slowed at reduced temperatures. Inhibitors of energy metabolism blocked the rate stimulation, as did cycloheximide; added nitrogen sources increased the rate additionally. The rate of sorbose uptake was also increased, whereas that of dimethylsulfoxide, which enters the cell by simple diffusion, was not changed. Transport of arabinose and fucose also occurred at increased rates. The data indicate a change in the sorbose transport system rather than in membrane permeability. The change, apparently the synthesis of a transport system component, requires metabolic energy and involves protein synthesis.     

Development of a helical-ribbon impeller bioreactor for high-density plant cell suspension culture

A double helical-ribbon impeller (HRI) bioreactor with a 11-L working volume was developed to grow high-density Catharanthus roseus cell suspensions. The rheological behavior of this suspension was found to be shear-thinning for concentrations higher than 12 to 15 g DW . L(-1). A granulated agar suspension of similar rheological properties was used as a model fluid for these suspensions. Mixing studies revealed that surface baffling and bottom profiling of the bioreactor and impeller speeds of 60 to 150 rpm ensured uniform mixing of suspensions. The HRI power requirement was found to increase singnificantly for agar suspensions higher than 13 g DW . L(-1), in conjunction with the effective viscosity increase. Oxygen transfer studies showed high apparent surface oxygen transfer coefficients (k(L)a approximately 4 to 45 h(-1)) from agar suspensions of 30 g DW . L(-1) to water and for mixing speeds ranging from 120 to 150 rpm. These high surface k(I)a values were ascribed to the flow pattern of this bioreactor configuration combined with surface bubble generation and entrainment in the liquid phase caused by the presence of the surface baffles. High-density C. roseus cell suspension cultures were successfully grown in this bioreactor without gas sparging. Up to 70% oxygen enrichment of the head space was required to ensure sufficient oxygen supply to the cultures so that dissolved oxygen concentration would remain above the critical level (>/=10% air saturation). The best mixing speed was 120 rpm. These cultures grew at the same rate ( approximately 0.4 d(-1)) and attained the same high biomass concentrations ( approximately 25 to 27 g DW . L(-1), 450 to 500 g filtered wet biomass . L(-1), and 92% to 100% settled wet biomass volume) as shake flask cultures. The scale-up potential of this bioreactor configuration is discussed.     

Imaging and manipulation of high-density lipoproteins.

The atomic force microscope (AFM) has been used to image a variety of biological systems, but has rarely been applied to soluble protein-lipid complexes. One of the primary physiological protein-lipid complexes is the high-density lipoproteins (HDL), responsible for the transport of cholesterol from the peripheral tissues and other lipoproteins to the liver. We have used the AFM to directly image discoidal reconstituted HDL (rHDL) particles for the first time. The height of these particles is consistent with a phospholipid bilayer structure, but careful high resolution measurements of particle diameters has indicated that they fuse when adsorbed to mica. Furthermore, it has been demonstrated that the AFM can be used to initiate this bilayer fusion in a controlled manner, allowing the fabrication of stabilized, nanometer scale, phospholipid bilayer "domains."     

CO2 in large-scale and high-density CHO cell perfusion culture

Productivity in a CHO perfusion culture reactor was maximized when pCO₂ was maintained in the range of 30–76 mm Hg. Higher levels of pCO₂ (> 150 mm Hg) resulted in CHO cell growth inhibition and dramatic reduction in productivity. We measured the oxygen utilization and CO₂ production rates for CHO cells in perfusion culture at 5.55×10^-17 mol cell^-1 sec^-1 and 5.36×10^-17 mol cell^-1 sec^-1 respectively. A simple method to directly measure the mass transfer coefficients for oxygen and carbon dioxide was also developed. For a 500 L bioreactor using pure oxygen sparge at 0.002 VVM from a microporous frit sparger, the overall apparent transfer rates (k_La+k_AA) for oxygen and carbon dioxide were 0.07264 min^-1 and 0.002962 min^-1 respectively. Thus, while a very low flow rate of pure oxygen microbubbles would be adequate to meet oxygen supply requirements for up to 2.1×10⁷ cells/mL, the low CO₂ removal efficiency would limit culture density to only 2.4×10⁶ cells/mL. An additional model was developed to predict the effect of bubble size on oxygen and CO₂ transfer rates. If pure oxygen is used in both the headspace and sparge, then the sparging rate can be minimized by the use of bubbles in the size range of 2–3 mm. For bubbles in this size range, the ratio of oxygen supply to carbon dioxide removal rates is matched to the ratio of metabolic oxygen utilization and carbon dioxide generation rates. Using this strategy in the 500 L reactor, we predict that dissolved oxygen and CO₂ levels can be maintained in the range to support maximum productivity (40% DO, 76 mm Hg pCO₂) for a culture at 10⁷ cells/mL, and with a minimum sparge rate of 0.006 vessel volumes per minute.A = volumetric agitated gas-liquid interfacial area at the top of the liquid, 1/mB = cell broth bleeding rate from the vessel, L/minCER = carbon dioxide evolution rate in the bioreactor, mol/min[CO₂] = dissolved CO₂ concentration in liquid, M[CO₂]^* = CO₂ concentration in equilibrium with sparger gas, M[CO₂]^** = CO₂ concentration in equilibrium with headspace gas, MCO₂(1) = dissolved carbon dioxide molecule in water[C_T] = total carbonic species concentration in bioreactor medium, M[C_T]_F = total carbonic species concentration in feed medium, MD = bioreactor diameter, mD_I = impeller diameter, mD_b = the initial delivered bubble diameter, mF = fresh medium feeding rate, L/minH_L = liquid height in the vessel, mk_A = carbon dioxide transfer coefficient at liquid surface, m/mink _infA ^supO = oxygen transfer coefficient at liquid surface, m/minNomenclature     

A binding protein-dependent transport system in Streptococcus mutans responsible for multiple sugar metabolism.

An 11-kilobase gene region of Streptococcus mutans has been identified which contains eight contiguous genes involved with the uptake and metabolism of multiple sugars (the msm system). Sequence analysis of this region indicates that several of these genes specify proteins with strong homology to components of periplasmic binding protein-dependent transport systems of Gram-negative bacteria. Additionally, this operon is controlled by a regulatory gene (msmR) that acts as a positive effector. The proteins specified by the structural genes of the msm operon include alpha-galactosidase (aga), a "periplasmic-like" sugar-binding protein (msmE), two membrane proteins (msmF, msmG), sucrose phosphorylase (gtfA), an ATP-binding protein (msmK), and dextran glucosidase (dexB). Insertional inactivation of each of these genes along with uptake data indicate that this system is responsible for the uptake of melibiose, raffinose, and isomaltotriose and the metabolism of melibiose, sucrose, and isomaltosaccharides.     

Potential of cell retention techniques for large-scale high-density perfusion culture of suspended mammalian cells

This review focuses on cultivation of mammalian cells in a suspended perfusion mode. The major technological limitation in the scaling-up of these systems is the need for robust retention devices to enable perfusion of medium as needed. For this, cell retention techniques available to date are presented, namely, cross-flow filters, hollow fibers, controlled-shear filters, vortex-flow filters, spin-filters, gravity settlers, centrifuges, acoustic settlers, and hydrocyclones. These retention techniques are compared and evaluated for their respective advantages and potential for large-scale utilization in the context of industrial manufacturing processes. This analysis shows certain techniques have a limited range of perfusion rate where they can be implemented (most microfiltration techniques). On the other hand, techniques were identified that have shown high perfusion capacity (centrifuges and spin-filters), or have a good potential for scale-up (acoustic settlers and inclined settlers). The literature clearly shows that reasonable solutions exist to develop large-scale perfusion processes.     

The nucellus: between cell elimination and sugar transport

The architecture of the seed is shaped by the processes of tissue partitioning, which determines the volume ratio of maternal and zygotic tissues, and nutrient partitioning, which regulates nutrient distribution among tissues. In angiosperms, early seed development is characterized by antagonistic development of the nucellus maternal tissue and the endosperm fertilization product to become the main sugar sink. This process marked the evolution of angiosperms and outlines the most ancient seed architectures. In Arabidopsis, the endosperm partially eliminates the nucellus and imports sugars from the seed coat. Here, we show that the nucellus is symplasmically connected to the chalaza, the seed nutrient unloading zone, and works as both a sugar sink and source alongside the seed coat. After fertilization, the transient nucellus accumulates starch early on and releases it in the apoplasmic space during its elimination. By contrast, the persistent nucellus exports sugars toward the endosperm through the SWEET4 hexose facilitator. Finally, we analyzed sugar metabolism and transport in the transparent testa 16 mutant, which fails to undergo nucellus cell elimination, which shed light on the coordination between tissue and nutrient partitioning. Overall, this study identifies a path of sugar transport in the Arabidopsis seed and describes a link between sugar redistribution and the nucellus cell-elimination program.

A path of sugar transport through the nucellus maternal tissue of the Arabidopsis seed is coordinated with the process of nucellus cell elimination.     

Cell metabolism measurements in culture via microelectronic biosensors

Serum free fermentation procedures of cell cultures have got a wide application in production of biochemicals. But, cells cultured in serum free media in general are more sensitive to changes in culture condition, especially to nutrient limitation. There are no substances from serum which can support the cells when conditions are changing. In this study special attention is directed to amino acid utilization of mouse hybridoma in batch, chemostat and perfusion fermentations. Detailed data are presented which show the considerable difference of amino acid consumption rates in different fermentation modes. Already, in batch mode there are differences of the two investigated mouse hybridoma cell lines, although they are derived from the same myeloma line. In chemostat running at a dilution rate representing maximal growth rate most of the consumption rates are significant higher than in batch. On the other hand, in perfusion mode the rates are lower than in batch. This indicates clearly the different conditions of the fermentation modes. Therefore, it is necessary to develop serum free processes under the desired production conditions. An accurate analysis of the process is strongly recommended.     

Proton movements coupled to sugar transport via the galactose transport system in Salmonella typhimurium.

We have studied proton movements associated with substrate transport via the galactose transport system in Salmonella typhimurium. The addition of galactose to lightly buffered suspensions of anaerobic, non-metabolizing cells of Salmonella typhimurium, specifically induced for the galactose transport system, causes an increase in extracellularpH as galactose and protons enter the cell together. Other substrates for this transport system, D-fucose, 2-deoxygalactose, glucose and 2-deoxyglucose similarly cause an influx of protons when transported. In contrast, transport via the other major transport system for galactose, the methylgalactoside transport system, is not coupled to H+ influx. Comparison of kinetic data obtained from pH measurements with data obtained from measurement of active transport of galactose via the galactose transport system suggests that the apparent Km of the galactose transport system for this sugar differs under energized and non-energized conditions. At pH 7.2 the permeant anion SCN- increases both the rate and extent of galactose-induced proton influx; at pH 6 the rate, but not the extent is increased by SCN-.     

Engineering of sugar metabolism of Corynebacterium glutamicum for production of amino acid l-alanine under oxygen deprivation

Corynebacterium glutamicum was genetically engineered to produce l-alanine from sugar under oxygen deprivation. The genes associated with production of organic acids in C. glutamicum were inactivated and the alanine dehydrogenase gene (alaD) from Lysinibacillus sphaericus was overexpressed to direct carbon flux from organic acids to alanine. Although the alaD-expressing strain produced alanine from glucose under oxygen deprivation, its productivity was relatively low due to retarded glucose consumption. Homologous overexpression of the gapA gene encoding glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in the alaD-expressing strain stimulated glucose consumption and consequently improved alanine productivity. In contrast gapA overexpression did not affect glucose consumption under aerobic conditions, indicating that oxygen deprivation engendered inefficient regeneration of NAD⁺ resulting in impaired GAPDH activity and reduced glucose consumption in the alanine-producing strains. Inactivation of the alanine racemase gene allowed production of l-alanine with optical purity greater than 99.5%. The resulting strain produced 98 g l⁻¹ of l-alanine after 32 h in mineral salts medium. Our results show promise for amino acid production under oxygen deprivation.