Expression of Genes Encoding F1-ATPase Results in Uncoupling of Glycolysis from Biomass Production in Lactococcus lactis

We studied how the introduction of an additional ATP-consuming reaction affects the metabolic fluxes in Lactococcus lactis. Genes encoding the hydrolytic part of the F₁ domain of the membrane-bound (F₁F₀) H⁺-ATPase were expressed from a range of synthetic constitutive promoters. Expression of the genes encoding F₁-ATPase was found to decrease the intracellular energy level and resulted in a decrease in the growth rate. The yield of biomass also decreased, which showed that the incorporated F₁-ATPase activity caused glycolysis to be uncoupled from biomass production. The increase in ATPase activity did not shift metabolism from homolactic to mixed-acid fermentation, which indicated that a low energy state is not the signal for such a change. The effect of uncoupled ATPase activity on the glycolytic flux depended on the growth conditions. The uncoupling stimulated the glycolytic flux threefold in nongrowing cells resuspended in buffer, but in steadily growing cells no increase in flux was observed. The latter result shows that glycolysis occurs close to its maximal capacity and indicates that control of the glycolytic flux under these conditions resides in the glycolytic reactions or in sugar transport.     

Co-factor engineering in lactobacilli: effects of uncoupled ATPase activity on metabolic fluxes in Lactobacillus (L.) plantarum and L. sakei

The hydrolytic F(1)-part of the F(1)F(0)-ATPase was over-expressed in Lactobacillus (L.) plantarum NC8 and L. sakei Lb790x during fermentation of glucose or ribose, in order to study how changes in the intracellular levels of ATP and ADP affect the metabolic fluxes. The uncoupled ATPase activity resulted in a decrease in intracellular energy level (ATP/ADP ratio), biomass yield and growth rate. Interestingly, the glycolytic and ribolytic flux increased in L. plantarum with uncoupled ATPase activity compared to the reference strain by up to 20% and 50%, respectively. The ATP demand was estimated to have approximately 80% control on both the glycolytic and ribolytic flux in L. plantarum under these conditions. In contrast, the glycolytic and ribolytic flux decreased in L. sakei with uncoupled ATPase activity.     

Experimental determination of control of glycolysis in Lactococcus lactis

The understanding of control of metabolic processes requires quantitative studies of the importance of the different enzymatic steps for the magnitude of metabolic fluxes and metabolite concentrations. An important element in such studies is the modulation of enzyme activities in small steps above and below the wild-type level. We review a genetic approach that is well suited for both Metabolic Optimization and Metabolic Control Analysis and studies on the importance of a number of glycolytic enzymes for metabolic fluxes in Lactococcus lactis. The glycolytic enzymes phosphofructokinase (PFK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PYK) and lactate dehydrogenase (LDH) are shown to have no significant control on the glycolytic flux in exponentially growing cells of L. lactis MG1363. Introduction of an uncoupled ATPase activity results in uncoupling of glycolysis from biomass production. With MG1363 growing in defined medium supplemented with glucose, the ATP demanding processes do not have a significant control on the glycolytic flux; it appears that glycolysis is running at maximal rate. It is likely that the flux control is distributed over many enzymes in L. lactis, but it cannot yet be excluded that one of the remaining glycolytic steps is a rate-limiting step for the glycolytic flux.     

The glycolytic flux in Escherichia coli is controlled by the demand for ATP

The nature of the control of glycolytic flux is one of the central, as-yet-uncharacterized issues in cellular metabolism. We developed a molecular genetic tool that specifically induces ATP hydrolysis in living cells without interfering with other aspects of metabolism. Genes encoding the F(1) part of the membrane-bound (F(1)F(0)) H(+)-ATP synthase were expressed in steadily growing Escherichia coli cells, which lowered the intracellular [ATP]/[ADP] ratio. This resulted in a strong stimulation of the specific glycolytic flux concomitant with a smaller decrease in the growth rate of the cells. By optimizing additional ATP hydrolysis, we increased the flux through glycolysis to 1.7 times that of the wild-type flux. The results demonstrate why attempts in the past to increase the glycolytic flux through overexpression of glycolytic enzymes have been unsuccessful: the majority of flux control (>75%) resides not inside but outside the pathway, i.e., with the enzymes that hydrolyze ATP. These data further allowed us to answer the question of whether catabolic or anabolic reactions control the growth of E. coli. We show that the majority of the control of growth rate resides in the anabolic reactions, i.e., the cells are mostly "carbon" limited. Ways to increase the efficiency and productivity of industrial fermentation processes are discussed.     

The extent to which ATP demand controls the glycolytic flux depends strongly on the organism and conditions for growth

Using molecular genetics we have introduced uncoupled ATPase activity in two different bacterial species, Escherichia coli and Lactococcus lactis, and determined the elasticities of the growth rate and glycolytic flux towards the intracellular [ATP]/[ADP] ratio. During balanced growth in batch cultures of E. coli the ATP demand was found to have almost full control on the glycolytic flux (FCC=0.96) and the flux could be stimulated by 70%. In contrast to this, in L. lactis the control by ATP demand on the glycolytic flux was close to zero. However, when we used non-growing cells of L. lactis (which have a low glycolytic flux) the ATP demand had a high flux control and the flux could be stimulated more than two fold. We suggest that the extent to which ATP demand controls the glycolytic flux depends on how much excess capacity of glycolysis is present in the cells.     

The role of ATPase in glycolysis of Ehrlich ascites tumor cells

Glycolysis in Ehrlich ascites tumor cells suspended in buffer containing 5 mM Pi was 50% inhibited by ouabain. In the absence of Pi the inhibition was less striking. Permeabilization of the cells with filipin abolished glycolysis, but glycolysis was restored by addition of Pi and AMP. Neither ouabain nor quercetin inhibited glycolysis in these permeabilized cells. We conclude that quercetin did not inhibit hexokinase sufficiently to affect glycolysis. An extract of Ehrlich ascites tumor cells glycolyzed weakly unless either Pi or an ATPase (e.g. (Na+K+)-ATPase) was added. The low rate of glycolysis of the extract was even further reduced when an endogenous ATPase was removed by precipitation with CaATP. The glycolytic activity of this ATPase-deficient extract was restored by addition of purified (Na+K+)-ATPase or of CaATP-precipitable ATPase. Addition of hexokinase without Pi did not restore glycolytic activity to the extract. An explanation for the contradictory conclusions by Bustamante, E., Morris, H.P., and Pedersen, P.L. (J. Biol. Chem. (1981) 265, 8699-8704) is presented.     

Increasing glycolytic flux in Torulopsis glabrata by redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation

AIMS: This study aimed at further increasing the pyruvate productivity of a multi-vitamin auxotrophic yeast Torulopsis glabrata by redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation. METHODS AND RESULTS: We examined two strategies to decrease the activity of F0F1-ATPase. The strategies were to inhibit F0F1-ATPase activity by addition of oligomycin, or to disrupt F0F1-ATPase by screening neomycin-resistant mutant. The addition of 0.05 mmol l(-1) oligomycin to the culture broth of T. glabrata CCTCC M202019 resulted in a significantly decreased intracellular ATP level (35.7%) and a significantly increased glucose consumption rate (49.7%). A neomycin-resistant mutant N07 was screened and selected after nitrosoguanidine mutagenesis of the parent strain T. glabrata CCTCC M202019. Compared with the parent strain, the F0F1-ATPase activity of the mutant N07 decreased about 65%. As a consequence, intracellular ATP level of the mutant N07 decreased by 24%, which resulted in a decreased growth rate and growth yield. As expected, glucose consumption rate and pyruvate productivity of the mutant N07 increased by 34% and 42.9%, respectively. Consistently, the activities of key glycolytic enzymes of the mutant N07, including phosphofructokinase, pyruvate kinase and glyceraldehyde-3-phosphate dehydrogenase, increased by 63.7%, 28.8% and 14.4%, respectively. In addition, activities of the key enzymes involved in electron transfer chain of the mutant N07 also increased. CONCLUSIONS: Impaired oxidative phosphorylation in T. glabrata leads to a decreased intracellular ATP production, thereby increasing the glycolytic flux. SIGNIFICANCE AND IMPACT OF THE STUDY: The strategy of redirecting ATP production from oxidative phosphorylation to substrate-level phosphorylation provides an alternative approach to enhance the glycolytic flux in eukaryotic micro-organisms.     

Use of the tac promoter and lacIq for the controlled expression of Zymomonas mobilis fermentative genes in Escherichia coli and Zymomonas mobilis.

The Zymomonas mobilis genes encoding alcohol dehydrogenase I (adhA), alcohol dehydrogenase II (adhB), and pyruvate decarboxylase (pdc) were overexpressed in Escherichia coli and Z. mobilis by using a broad-host-range vector containing the tac promoter and the lacIq repressor gene. Maximal IPTG (isopropyl-beta-D-thiogalactopyranoside) induction of these plasmid-borne genes in Z. mobilis resulted in a 35-fold increase in alcohol dehydrogenase I activity, a 16.7-fold increase in alcohol dehydrogenase II activity, and a 6.3-fold increase in pyruvate decarboxylase activity. Small changes in the activities of these enzymes did not affect glycolytic flux in cells which are at maximal metabolic activity, indicating that flux under these conditions is controlled at some other point in metabolism. Expression of adhA, adhB, or pdc at high specific activities (above 8 IU/mg of cell protein) resulted in a decrease in glycolytic flux (negative flux control coefficients), which was most pronounced for pyruvate decarboxylase. Growth rate and flux are imperfectly coupled in this organism. Neither a twofold increase in flux nor a 50% decline from maximal flux caused any immediate change in growth rate. Thus, the rates of biosynthesis and growth in this organism are not limited by energy generation in rich medium.     

Glycolytic flux is conditionally correlated with ATP concentration in Saccharomyces cerevisiae: a chemostat study under carbon- or nitrogen-limiting conditions.

Anaerobic and aerobic chemostat cultures of Saccharomyces cerevisiae were performed at a constant dilution rate of 0.10 h(-1). The glucose concentration was kept constant, whereas the nitrogen concentration was gradually decreasing; i.e., the conditions were changed from glucose and energy limitation to nitrogen limitation and energy excess. This experimental setup enabled the glycolytic rate to be separated from the growth rate. There was an extensive uncoupling between anabolic energy requirements and catabolic energy production when the energy source was present in excess both aerobically and anaerobically. To increase the catabolic activity even further, experiments were carried out in the presence of 5 mM acetic acid or benzoic acid. However, there was almost no effect with acetate addition, whereas both respiratory (aerobically) and fermentative activities were elevated in the presence of benzoic acid. There was a strong negative correlation between glycolytic flux and intracellular ATP content; i.e., the higher the ATP content, the lower the rate of glycolysis. No correlation could be found with the other nucleotides tested (ADP, GTP, and UTP) or with the ATP/ADP ratio. Furthermore, a higher rate of glycolysis was not accompanied by an increasing level of glycolytic enzymes. On the contrary, the glycolytic enzymes decreased with increasing flux. The most pronounced reduction was obtained for HXK2 and ENO1. There was also a correlation between the extent of carbohydrate accumulation and glycolytic flux. A high accumulation was obtained at low glycolytic rates under glucose limitation, whereas nitrogen limitation during conditions of excess carbon and energy resulted in more or less complete depletion of intracellular storage carbohydrates irrespective of anaerobic or aerobic conditions. However, there was one difference in that glycogen dominated anaerobically whereas under aerobic conditions, trehalose was the major carbohydrate accumulated. Possible mechanisms which may explain the strong correlation between glycolytic flux, storage carbohydrate accumulation, and ATP concentrations are discussed.     

Effects of overexpression of phosphofructokinase on glycolysis in the yeast Saccharomyces cerevisiae.

The influence of 6-phosphofructo-1-kinase on glycolytic flux in the yeast Saccharomyces cerevisiae was assessed by measuring the effects of enzyme overexpression on glucose consumption, ethanol production, and glycolytic intermediate levels under aerobic and anaerobic conditions. Enzyme overexpression had no effect on glycolytic flux under anaerobic conditions, but under aerobic conditions, it increased glycolytic flux up to the anaerobic level. The Pasteur effect was thus abolished in these cells. The increased glycolytic flux was accompanied by a compensatory decrease in flux in oxidative phosphorylation. The concentrations of the enzyme substrates showed only small or insignificant changes. These data imply that the enzyme has a low flux control coefficient for glycolysis. However, in cells overexpressing the enzyme, there was a compensatory decrease in 6-phosphofructo-2-kinase activity which was accompanied by a corresponding decrease in fructose 2,6-bisphosphate concentration. Measurements in vitro showed that the decrease in the concentration of this positive allosteric effector of 6-phosphofructo-1-kinase could significantly lower its specific activity in the cell and that this could compensate for the increased enzyme concentration in the overproducer.     

NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na(+)-K(+)-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na(+)-K(+)-ATPase by 10 microM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H(+) transporter secondary to decreased availability of H(+) for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H(+) transporter, which was specifically linked to reduced activity of Na(+)-K(+)-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na(+)-K(+)-ATPase.     

Diethylstilbestrol. A novel F0-directed probe of the mitochondrial proton ATPase

At low concentrations, diethylstilbestrol (DES) is shown to be a potent F0-directed inhibitor of the F0F1-ATPase of rat liver mitochondria. In analogy to other F0-directed inhibitors, DES inhibits both the ATPase and ATP-dependent proton-translocation activities of the purified and membrane bound enzyme. When added at low concentrations with dicyclohexylcarbodiimide (DCCD), a covalent inhibitor, DES acts synergistically to inhibit ATPase activity of the complex. At higher concentrations, DES restores DCCD-inhibited ATPase activity. However, there is no restoration of ATP-dependent proton translocation. Under these conditions DCCD remains covalently bound to the F0F1-ATPase complex and F1 remains bound to Fo. Significantly, when the F0F1-ATPase is inhibited by the Fo-directed inhibitor venturicidin rather than DCCD, DES is also able to restore ATPase activity. In contrast, DES is unable to restore ATPase activity to F0F1 preparations inhibited by the Fo-directed inhibitors oligomycin or tricyclohexyltin. However, combinations of [DES + DCCD] or [DES + venturicidin] can restore ATPase activity to F0F1 preparations inhibited by either oligomycin or tricyclohexyltin. Results presented here indicate that the F0 moiety of the rat liver mitochondrial proton ATPase contains a distinct binding site for DES. In addition, they suggest that at saturating concentrations simultaneous occupancy of the DES binding site and sites for either DCCD or venturicidin promote "uncoupled" ATP hydrolysis.     

Increasing acidification of nonreplicating Lactococcus lactis deltathyA mutants by incorporating ATPase activity

Lactococcus lactis MBP71 deltathyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis.     

The study of the pathogenic mechanism of mitochondrial diseases provides information on basic bioenergetics

Mitochondrial F(1)F(0)-ATPase was studied in lymphocytes from patients with neuropathy, ataxia, and retinitis pigmentosa (NARP), caused by a mutation at leu-156 in the ATPase 6 subunit. The mutation giving the milder phenotype (Leu156Pro) suffered a 30% reduction in proton flux, and a similar loss in ATP synthetic activity. The more severe mutation (Leu156Arg) also suffered a 30% reduction in proton flux, but ATP synthesis was virtually abolished. Oligomycin sensitivity of the proton translocation through F(0) was enhanced by both mutations. We conclude that in the Leu156Pro mutation, rotation of the c-ring is slowed but coupling of ATP synthesis to proton flux is maintained, whereas in the Leu156Arg mutation, proton flux appears to be uncoupled. Modelling indicated that, in the Leu156Arg mutation, transmembrane helix III of ATPase 6 is unable to span the membrane, terminating in an intramembrane helix II-helix III loop. We propose that the integrity of transmembrane helix III is essential for the mechanical function of ATPase 6 as a stator element in the ATP synthase, but that it is not relevant for oligomycin inhibition.     

Energy metabolism transition in multi-cellular human tumor spheroids

It is thought that glycolysis is the predominant energy pathway in cancer, particularly in solid and poorly vascularized tumors where hypoxic regions develop. To evaluate whether glycolysis does effectively predominate for ATP supply and to identify the underlying biochemical mechanisms, the glycolytic and oxidative phosphorylation (OxPhos) fluxes, ATP/ADP ratio, phosphorylation potential, and expression and activity of relevant energy metabolism enzymes were determined in multi-cellular tumor spheroids, as a model of human solid tumors. In HeLa and Hek293 young-spheroids, the OxPhos flux and cytochrome c oxidase protein content and activity were similar to those observed in monolayer cultured cells, whereas the glycolytic flux increased two- to fourfold; the contribution of OxPhos to ATP supply was 60%. In contrast, in old-spheroids, OxPhos, ATP content, ATP/ADP ratio, and phosphorylation potential diminished 50-70%, as well as the activity (88%) and content (3 times) of cytochrome c oxidase. Glycolysis and hexokinase increased significantly (both, 4 times); consequently glycolysis was the predominant pathway for ATP supply (80%). These changes were associated with an increase (3.3 times) in the HIF-1alpha content. After chronic exposure, both oxidative and glycolytic inhibitors blocked spheroid growth, although the glycolytic inhibitors, 2-deoxyglucose and gossypol (IC(50) of 15-17 nM), were more potent than the mitochondrial inhibitors, casiopeina II-gly, laherradurin, and rhodamine 123 (IC(50) > 100 nM). These results suggest that glycolysis and OxPhos might be considered as metabolic targets to diminish cellular proliferation in poorly vascularized, hypoxic solid tumors.     

Role of F1F0-ATPase in the growth of streptococcus mutans GS5

The role of F1F0-ATPase in Streptococcus mutans GS5 was investigated by isolating a mutant (NTS1) defective in enzyme activity by homologous recombination with a plasmid encoding the 5' terminal fragment of the F1F0-ATPase beta-subunit gene. The ATPase activity of NTS1 membranes was 49% that of GS5 membranes. The lag phase of the growth curve of NTS1 was longer than that for GS5, and the lag phase of GS5 and NTS1 were prolonged by the addition of ionophore gramicidin D; at stationary phase, the turbidity of the NTS1 culture was less than that of the GS5 culture. These results suggest that S. mutans F1F0-ATPase contributes to the generation of a stoichiometric electrogenic gradient effectively in the lag phase.     

Increasing Acidification of Nonreplicating Lactococcus lactis ΔthyA Mutants by Incorporating ATPase Activity

Lactococcus lactis MBP71 ΔthyA (thymidylate synthase) cannot synthesize dTTP de novo, and DNA replication is dependent on thymidine in the growth medium. In the nonreplicating state acidification by MBP71 was completely insensitive to bacteriophages (M. B. Pedersen, P. R. Jensen, T. Janzen, and D. Nilsson, Appl. Environ. Microbiol. 68:3010-3023, 2002). For nonreplicating MBP71 the biomass increased 3.3-fold over the first 3.5 h, and then the increase stopped. The rate of acidification increased 2.3-fold and then started to decrease. Shortly after inoculation the lactic acid flux was 60% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by nonreplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis.