Modification of central metabolic pathway in escherichia coli to reduce acetate accumulation by heterologous expression of the bacillus subtilis acetolactate synthase gene

A novel metabolic engineering technique involving the redirection ofcellular carbon fluxes was employed to reduce acetate production in an Escherichia coli culture. Metabolic engineering was achieved by cloning E. coli the gene for the Bacillus subtilis acetolactate synthase (ALS), an enzyme capable of catalyzing the conversion of pyruvate to nonacidic and less harmful species. The heterologous expression of the ALS catabolic enzyme in Escherichia coli drastically modified the cellular glycolytic fluxes. In particular, acetate excretion, which is a common characteristic of E. coli, as well as a physiological burden, was minimized. The residual acetate level was kept under control and maintained at a level that was below the toxic threshold. The expression of the biologically active ALS enzyme in E. coli did not result in any detectable changes on either cell growth rate or cell yields. The alternative product, acetoin, was shown to be 50 times less harmful than acetate. Similarities in the growth pattern of two different E. coli strains, RR1 and GJT001, under all cultivation conditions suggested that the ability of ALS to reduce acetate accumulation is generic and not strain-specific. (c) 1994 John Wiley & Sons, Inc.     

Metabolic flux analysis of Escherichia coli deficient in the acetate production pathway and expressing the Bacillus subtilis acetolactate synthase

Several approaches to reduce acetate accumulation in Escherichia coli cultures have recently been reported. This reduction subsequently led to a significant enhancement in recombinant protein production. In those studies, metabolically engineered E. coli strains with reduced acetate synthesis rates were constructed through the modification of glucose uptake rate, the elimination of critical enzymes that are involved in the acetate formation pathways, and the redirection of carbon flux toward less inhibitory byproducts. In particular, it has been shown that strains carrying the Bacillus subtilis acetolactate synthase (ALS) gene not only produce less acetate but also have a higher ATP yield. Metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point revealed that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin. The main focus of this study is the systematic analysis of the effects of small perturbations in the host's existing pathways on the redistribution of carbon fluxes. Specifically, a mutant with deleted acetate kinase (ACK) and acetyl phosphotransferase (PTA) was constructed and studied. Results from the metabolic analysis of carbon redistribution show the ackA-pta mutation will reduce acetate level at the expense of the growth rate. In addition, in the ackA-pta deficient strain a much higher lactate formation rate with simultaneously lower formate and ethanol synthesis rates was found. Expression of the B. subtilis ALS in ackA-pta mutants further reduces acetate levels while cell density similar to that of the parent strain is attained.     

Systematic identification and elimination of flux bottlenecks in the aldehyde production pathway of Synechococcus elongatus PCC 7942

Isotopically nonstationary metabolic flux analysis (INST-MFA) provides a versatile platform to quantitatively assess in vivo metabolic activities of autotrophic systems. By applying INST-MFA to recombinant aldehyde-producing cyanobacteria, we identified metabolic alterations that correlated with increased strain performance in order to guide rational metabolic engineering. We identified four reactions adjacent to the pyruvate node that varied significantly with increasing aldehyde production: pyruvate kinase (PK) and acetolactate synthase (ALS) fluxes were directly correlated with product formation, while pyruvate dehydrogenase (PDH) and phosphoenolpyruvate carboxylase (PPC) fluxes were inversely correlated. Overexpression of enzymes for PK or ALS did not result in further improvements to the previous best-performing strain, while downregulation of PDH expression (through antisense RNA expression) or PPC flux (through expression of the reverse reaction, phosphoenolpyruvate carboxykinase) provided significant improvements. These results illustrate the potential of INST-MFA to enable a systematic approach for iterative identification and removal of pathway bottlenecks in autotrophic host cells.     

Comparison of the omega-transaminases from different microorganisms and application to production of chiral amines

Microorganisms that are capable of (S)-enantioselective transamination of chiral amines were isolated from soil samples by selective enrichment using (S)-alpha-methyl-benzylamine ((S)-alpha-MBA) as a sole nitrogen source. Among them, Klebsiella pneumoniae JS2F, Bacillus thuringiensis JS64, and Vibrio fluvialis JS17 showed good omega-transaminase (omega-TA) activities and the properties of the omega-TAs were investigated. The induction level of the enzyme was strongly dependent on the nitrogen source for the strains, except for V. fluvialis JS17. All the omega-TAs showed high enantioselectivity (E>50) toward (S)-alpha-MBA and broad amino donor specificities for arylic and aliphatic chiral amines. Besides pyruvate, aldehydes such as propionaldehyde and butyraldehyde showed good amino acceptor reactivities. All the omega-TAs showed substrate inhibition by (S)-alpha-MBA above 200 mm. Moreover, substrate inhibition by pyruvate above 10 mm was observed for omega-TA from V. fluvialis JS17. In the case of product inhibition, acetophenone showed much greater inhibitions than L-alanine for all omega-TAs. Comparison of the enzyme properties indicates that omega-transaminase from V. fluvialis JS17 is the best one for both kinetic resolution and asymmetric synthesis to produce enantiomerically pure chiral amines. Kinetic resolution of sec-butylamine (20 mM) was done under reduced pressure (150 Torr) to selectively remove an inhibitory product (2-butanone) using the enzyme from V. fluvialis JS17. Enantiomeric excess of (R)-sec-butylamine reached 94.7% after 12 h of reaction.     

omega-Amino acid:pyruvate transaminase from Alcaligenes denitrificans Y2k-2: a new catalyst for kinetic resolution of beta-amino acids and amines

Alcaligenes denitrificans Y2k-2 was obtained by selective enrichment followed by screening from soil samples, which showed omega-amino acid:pyruvate transaminase activity, to kinetically resolve aliphatic beta-amino acid, and the corresponding structural gene (aptA) was cloned. The gene was functionally expressed in Escherichia coli BL21 by using an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible pET expression system (9.6 U/mg), and the recombinant AptA was purified to show a specific activity of 77.2 U/mg for L-beta-amino-n-butyric acid (L-beta-ABA). The enzyme converts various beta-amino acids and amines to the corresponding beta-keto acids and ketones by using pyruvate as an amine acceptor. The apparent K(m) and V(max) for L-beta-ABA were 56 mM and 500 U/mg, respectively, in the presence of 10 mM pyruvate. In the presence of 10 mM L-beta-ABA, the apparent K(m) and V(max) for pyruvate were 11 mM and 370 U/mg, respectively. The enzyme exhibits high stereoselectivity (E > 80) in the kinetic resolution of 50 mM D,L-beta-ABA, producing optically pure D-beta-ABA (99% enantiomeric excess) with 53% conversion.     

Effect of a pyruvate kinase (pykF-gene) knockout mutation on the control of gene expression and metabolic fluxes in Escherichia coli

The metabolic regulation of Escherichia coli lacking a functional pykF gene was investigated based on gene expressions, enzyme activities, intracellular metabolite concentrations and the metabolic flux distribution obtained based on (13)C-labeling experiments. RT-PCR revealed that the glycolytic genes such as glk, pgi, pfkA and tpiA were down regulated, that ppc, pckA, maeB and mdh genes were strongly up-regulated, and that the oxidative pentose phosphate pathway genes such as zwf and gnd were significantly up-regulated in the pykF mutant. The catabolite repressor/activator gene fruR was up-regulated in the pykF mutant, but the adenylate cyclase gene cyaA was down-regulated indicating a decreased rate of glucose uptake. This was also ascertained by the degradation of ptsG mRNA, the gene for which was down-regulated in the pykF mutant. In general, the changes in enzyme activities more or less correlated with ratios of gene expression, while the changes in metabolic fluxes did not correlate with enzyme activities. For example, high flux ratios were obtained through the oxidative pentose phosphate pathway due to an increased concentration of glucose-6-phosphate rather than to favorable enzyme activity ratios. In contrast, due to decreased availability of pyruvate (and acetyl coenzyme A) in the pykF mutant compared with the wild type, low flux ratios were found through lactate and acetate forming pathways.     

Biochemical and molecular characterization of taurine:pyruvate aminotransferase from the anaerobe Bilophila wadsworthia.

Bilophila wadsworthia RZATAU is a Gram-negative bacterium which converts the sulfonate taurine (2-aminoethanesulfonate) to ammonia, acetate and sulfide in an anaerobic respiration. Taurine:pyruvate aminotransferase (Tpa) catalyses the initial metabolic reaction yielding alanine and sulfoacetaldehyde. We purified Tpa 72-fold to apparent homogeneity with an overall yield of 89%. The purified enzyme did not require addition of pyridoxal 5'-phosphate, but highly active enzyme was only obtained by addition of pyridoxal 5'-phosphate to all buffers during purification. SDS/PAGE revealed a single protein band with a molecular mass of 51 kDa. The apparent molecular mass of the native enzyme was 197 kDa as determined by gel filtration, which indicates a homotetrameric structure. The kinetic constants for taurine were: Km = 7.1 mM, Vmax = 1.20 nmol.s-1, and for pyruvate: Km = 0.82 mM, Vmax = 0.17 nmol.s-1. The purified enzyme was able to transaminate hypotaurine (2-aminosulfinate), taurine, beta-alanine and with low activity cysteine and 3-aminopropanesulfonate. In addition to pyruvate, 2-ketobutyrate and oxaloacetate were utilized as amino group acceptors. We have sequenced the encoding gene (tpa). It encoded a 50-kDa peptide, which revealed 33% identity to diaminopelargonate aminotransferase from Bacillus subtilis.     

Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture: II. Analysis of metabolic rates and pathways under oscillation and steady-state conditions

The oscillation phenomena reported in the preceding article for the anaerobic continuous fermentation of glycerol by Klebsiella pneumoniae are analyzed in terms of metabolic fluxes (metabolic rates and yields) and stoichiometry of pathways. Significant oscillations in the fluxes of CO(2), H(2), formic acid, ethanol, and reducing equivalents are observed which show obvious relationships to each other. Changes in the consumption or production rates of glycerol, acetic acid, 1,3-propanediol, and ATP are irregular and have relatively small amplitudes compared with their absolute values. By comparing the metabolic fluxes under oscillation and steady state that have nearly the same environmental conditions it could be shown that pyruvate metabolism is the main step affected under oscillation conditions. The specific formation rates of all the products originating from pyruvate metabolism (CO(2), H(2), formic acid, ethanol, acetic acid, lactic acid, and 2,3-butanediol) show significant differences under conditions of oscillation and steady state. In contrast, the specific rates of substrate uptake, ATP generation, and formation of products deriving either directly from glycerol (1,3-propanediol) or from the upstream of pyruvate metabolism (e.g., succinic acid) are not, or at least not significantly, affected during oscillation. Stoichiometric analysis of metabolic pathways confirms that other enzyme systems, in addition to pyruvate: formate-lyase, must be simultaneously involved in the pyruvate decarboxylation under both oscillation and steady-state conditions. The results strongly suggest oscillations of activities of these enzymes under oscillation conditions. It appears that the reason for the occurrence of oscillation and hysteresis lies in an unstable regulation of pyruvate metabolism of different enzymes triggered by substrate excess and drastic change(s) of environmental conditions. (c) 1996 John Wiley & Sons, Inc.     

The crystal structures of Klebsiella pneumoniae acetolactate synthase with enzyme-bound cofactor and with an unusual intermediate

Acetohydroxyacid synthase (AHAS) and acetolactate synthase (ALS) are thiamine diphosphate (ThDP)-dependent enzymes that catalyze the decarboxylation of pyruvate to give a cofactor-bound hydroxyethyl group, which is transferred to a second molecule of pyruvate to give 2-acetolactate. AHAS is found in plants, fungi, and bacteria, is involved in the biosynthesis of the branched-chain amino acids, and contains non-catalytic FAD. ALS is found only in some bacteria, is a catabolic enzyme required for the butanediol fermentation, and does not contain FAD. Here we report the 2.3-A crystal structure of Klebsiella pneumoniae ALS. The overall structure is similar to AHAS except for a groove that accommodates FAD in AHAS, which is filled with amino acid side chains in ALS. The ThDP cofactor has an unusual conformation that is unprecedented among the 26 known three-dimensional structures of nine ThDP-dependent enzymes, including AHAS. This conformation suggests a novel mechanism for ALS. A second structure, at 2.0 A, is described in which the enzyme is trapped halfway through the catalytic cycle so that it contains the hydroxyethyl intermediate bound to ThDP. The cofactor has a tricyclic structure that has not been observed previously in any ThDP-dependent enzyme, although similar structures are well known for free thiamine. This structure is consistent with our proposed mechanism and probably results from an intramolecular proton transfer within a tricyclic carbanion that is the true reaction intermediate. Modeling of the second molecule of pyruvate into the active site of the enzyme with the bound intermediate is consistent with the stereochemistry and specificity of ALS.     

Characterization of the fructose 1,6-bisphosphate-activated, L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus.

The L(+)-lactate dehydrogenase from Thermoanaerobacter ethanolicus wt was purified to a final specific activity of 598 mumol pyruvate reduced per min per mg of protein. The specific activity of the pure enzyme with L(+)-lactate was 0.79 units per mg of protein. The M(r) of the native enzyme was 134,000 containing a single subunit type of M(r) 33,500 indicating an apparent tetrameric structure. The L(+)-lactate dehydrogenase was activated by fructose 1,6-bisphosphate in a cooperative manner affecting Vmax and Km values. The activity of the enzyme was also effected by pH, pyruvate and NADH. The Km for NADH at pH 6.0 was 0.05 mM and the Vmax for pyruvate reduction at pH 6.0 was 1082 units per mg in the presence of 1 mM fructose 1,6-bisphosphate. The enzyme was inhibited by NADPH, displaying an uncompetitive pattern. This pattern indicated that NADPH was a negative modifier of the enzyme. The role of L(+)-lactate dehydrogenase in controlling the end products of fermentation is discussed.     

Global metabolic response of Escherichia coli to gnd or zwf gene-knockout, based on 13C-labeling experiments and the measurement of enzyme activities

An integrated study on cell growth, enzyme activities and carbon flux redistribution was made to investigate how the central metabolism of Escherichia coli changes with the knockout of genes in the oxidative pentose phosphate pathway (PPP). Mutants deficient in glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were constructed by disrupting the zwf and gnd genes and were grown in minimal media with two different carbon sources, such as glucose or pyruvate. It was shown that the knockout of either gnd or zwf gene did not affect the cell growth rate significantly, but the cellular metabolism was changed. While the specific substrate uptake rate and the specific carbon dioxide evolution rate for either mutant grown on glucose were higher than those obtained for the parent strain, these two rates were markedly decreased in mutants grown on pyruvate. The measurement of enzyme activities implied a significant change in metabolism, when alternative pathways such as the Entner–Doudoroff pathway (EDP) and the malic enzyme pathway were activated in the gnd mutant grown on glucose. As compared with the parent strain, the activities of phosphoglucose isomerase were increased in mutants grown on glucose but decreased in mutants grown on pyruvate. The metabolic flux redistribution obtained based on ¹³C-labeling experiments further indicated that the direction of the flux through the non-oxidative PPP was reversed in response to the gene knockout. Moreover, the knockout of genes caused an increased flux through the tricarboxlic acid cycle in mutants grown on glucose but caused a decrease in the case of using pyruvate. There was also a negative correlation between the fluxes through malic enzyme and isocitrate dehydrogenase in the mutants; and a positive correlation was found between the fluxes through malic enzyme and phosphoenolpyruvate carboxylase.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Carbon sources affect metabolic capacities of Bacillus species for the production of industrial enzymes: theoretical analyses for serine and neutral proteases and alpha-amylase

The metabolic fluxes through the central carbon pathways were calculated for the genus Bacillus separately for the enzymes serine alkaline protease (SAP), neutral protease (NP) and alpha-amylase (AMY) on five carbon sources that have different reduction degrees (gamma), to determine the theoretical ultimate limits of the production capacities of Bacillus species and to predict the selective substrate for the media design. Glucose (gamma=4.0), acetate (gamma=4.0), and the TCA cycle organic-acids succinate (gamma=3.5), malate (gamma=3.0), and citrate (gamma=3.0) were selected for the theoretical analyses and comparisons. A detailed mass flux balance-based general stoichiometric model based on the proposed metabolic reaction network starting with the alternative five carbon sources for the synthesis of each enzyme in Bacillus licheniformis that simulates the behaviour of the metabolic pathways with 107 metabolites and 150 reaction fluxes is developed. Highest and lowest specific cell growth rates (&mgr;) were calculated as 1.142 and 0.766h(-1), respectively, when glucose that has the highest degree of reduction and citrate that has the lowest degree of reduction were used as the carbon sources. Highest and lowest SAP, NP and AMY synthesis rates were also obtained, respectively, when glucose and citrate were used. Metabolic capacity analyses showed that the maximum SAP, NP, and AMY synthesis rates were, respectively, 0.0483, 0.0215 and 0.0191mmolg(-1)DWh(-1) when glucose uptake rate was 10mmolg(-1)DWh(-1) and specific growth rate was zero. The amino acid compositions and the molecular weights of the enzyme influence the production yield and selectivity. For SAP and NP oxaloacetate and pyruvate, for AMY oxaloacetate appear to be the critical main branch points. Consequently, for SAP and NP syntheses the fluxes towards the alanine group and aspartate group, and for AMY synthesis the flux towards the aspartate group amino acids need to be high. The results encourage the discussion of the potential strategies for improving productions of SAP, NP and AMY.     

A synthesis of acylphosphonic acids and of 1-aminoalkylphosphonic acids: the action of pyruvate dehydrogenase and lactate dehydrogenase on acetylphosphonic acid.

Acylphosphonic acids, R-CO-PO(OH)2, have been synthesized by the steps [formula: see text] of which the last is new and provides a mild method for de-esterifying acylphosphonic acids. Their reductive amination gives a simple way of making 1-aminoalkylphosphonic acids. Acetylphosphonic acid inhibited NAD+ reduction by pyruvate with the pyruvate dehydrogenases from Escherichia coli and Bacillus stearothermophilus. The inhibition was competitive with pyruvate, with Ki of 6 microM for the E. coli enzyme (pyruvate Km 0.5 mM) and one of 0.4 mM of the B. stearothermophilus enzyme (pyruvate Km 0.1 mM). Acetylphosphonate and its monomethyl ester are substates for pig heart lactate dehydrogenase, with Km values of 15 mM and 10 mM respectively (pyruvate Km 0.05 mM) and specificity constants one thousandth that for pyruvate.     

The effects of alpha-adrenergic stimulation on the regulation of the pyruvate dehydrogenase complex in the perfused rat liver

The regulation of the pyruvate dehydrogenase multienzyme complex was investigated during alpha-adrenergic stimulation with phenylephrine in the isolated perfused rat liver. The metabolic flux through the pyruvate dehydrogenase reaction was monitored by measuring the production of 14CO2 from infused [1-14C] pyruvate. In livers from fed animals perfused with a low concentration of pyruvate (0.05 mM), phenylephrine infusion significantly inhibited the rate of pyruvate decarboxylation without affecting the amount of pyruvate dehydrogenase in its active form. Also, phenylephrine caused no significant effect on tissue NADH/NAD+ and acetyl-CoA/CoASH ratios or on the kinetics of pyruvate decarboxylation in 14CO2 washout experiments. Phenylephrine inhibition of [1-14C]pyruvate decarboxylation was, however, closely associated with a decrease in the specific radioactivity of perfusate lactate, suggesting that the pyruvate decarboxylation response simply reflected dilution of the labeled pyruvate pool due to phenylephrine-stimulated glycogenolysis. This suggestion was confirmed in additional experiments which showed that the alpha-adrenergic-mediated inhibitory effect on pyruvate decarboxylation was reduced in livers perfused with a high concentration of pyruvate (1 mM) and was absent in livers from starved rats. Thus, alpha-adrenergic agonists do not exert short term regulatory effects on pyruvate dehydrogenase in the liver. Furthermore, the results suggest either that the rat liver pyruvate dehydrogenase complex is insensitive to changes in mitochondrial calcium or that changes in intramitochondrial calcium levels as a result of alpha-adrenergic stimulation are considerably less than suggested by others.     

Metabolic flux analysis of Escherichia coli expressing the Bacillus subtilis acetolactate synthase in batch and continuous cultures.

Metabolically engineered Escherichia coli expressing the B. subtilis acetolactate synthase has shown to be capable of reducing acetate accumulation. This reduction subsequently led to a significant enhancement in recombinant protein production. The main focus of this study is to systematically examine the effect of ALS in the metabolic patterns of E. coli in batch and continuous culture. The specific acetate production rate of a strain carrying the B. subtilis als gene is 75% lower than that of the control strain (host carrying the control plasmid pACYC184) in batch cultures. The ALS strain is further demonstrated to be capable of maintaining a reduced specific acetate production rate in continuous cultures at dilution rates ranging from 0.1 to 0.4 h-1. In addition, this ALS strain is shown to have a higher ATP yield and lower maintenance coefficient. The metabolic flux analysis of carbon flux distribution of the central metabolic pathways and at the pyruvate branch point reveals that this strain has the ability to channel excess pyruvate to the much less toxic compound, acetoin.     

Effect of inactivation of nuo and ackA-pta on redistribution of metabolic fluxes in Escherichia coli.

The nuoA-N gene cluster encodes a transmembrane NADH:ubiquinone oxidoreductase (NDH-I) responsible for coupling redox chemistry to proton-motive force generation. Interactions between nuo and the acetate-producing pathway encoded by ackA-pta were investigated by examining the metabolic patterns of several mutant strains under anaerobic growth conditions. In an ackA-pta strain, the flux to acetate was decreased dramatically, whereas flux to lactate was increased significantly when compared with its parent strain; the fluxes to pyruvate and ethanol also increased slightly. In addition, pyruvate was excreted. A strain carrying the nuo mutation showed metabolic flux distribution similar to the wild type. The ackA-pta-nuo strain showed a different metabolic pattern. It not only exhibited reduced acetate accumulation but also significantly lower ethanol and formate synthesis. Metabolic flux distribution analysis suggests that the excessive carbon flux was redirected at the pyruvate node through the lactate dehydrogenase pathway for lactate formation rather than the pyruvate formate-lyase (PFL) pathway for acetyl-CoA and formate production. The diminished capacity through the formate and ethanol (ADH) pathways was not the result of genetic disruption of functional PFL or ADH production. The introduction of a Bacillus subtilis acetolactate synthase gene returned formate, ethanol, and lactate levels to those of the wild type (ackA(+)pta(+)nuo(+)) strain. Furthermore, transfer of a lactate dehydrogenase mutation yielded a strain producing ethanol as the sole fermentation product. As confirmation of the nuo effect, cultures of the ackA-pta strain, supplemented with an NDH-I inhibitor, produced intermediary levels of flux to ethanol and formate. Mutations in both ackA-pta and nuo are required to significantly reduce the flux through the PFL pathway.     

Coordinated reprogramming of metabolism and cell function in adipocytes from proliferation to differentiation

Adipose tissue plays a major role in regulating lipid and energy homeostasis by storing excess nutrients, releasing energetic substrates through lipolysis, and regulating metabolism of other tissues and organs through endocrine and paracrine signaling. Adipocytes within fat tissues store excess nutrients through increased cell number (hyperplasia), increased cell size (hypertrophy), or both. The differentiation of pre-adipocytes into mature lipid-accumulating adipocytes requires a complex interaction of metabolic pathways that is still incompletely understood. Here, we applied parallel labeling experiments and ¹³C-metabolic flux analysis to quantify precise metabolic fluxes in proliferating and differentiated 3T3-L1 cells, a widely used model to study adipogenesis. We found that morphological and biomass composition changes in adipocytes were accompanied by significant shifts in metabolic fluxes, encompassing all major metabolic pathways. In contrast to proliferating cells, differentiated adipocytes 1) increased glucose uptake and redirected glucose utilization from lactate production to lipogenesis and energy generation; 2) increased pathway fluxes through glycolysis, oxidative pentose phosphate pathway and citric acid cycle; 3) reduced lactate secretion, resulting in increased ATP generation via oxidative phosphorylation; 4) rewired glutamine metabolism, from glutaminolysis to de novo glutamine synthesis; 5) increased cytosolic NADPH production, driven mostly by increased cytosolic malic enzyme flux; 6) increased production of monounsaturated C16:1; and 7) activated a mitochondrial pyruvate cycle through simultaneous activity of pyruvate carboxylase, malate dehydrogenase and malic enzyme. Taken together, these results quantitatively highlight the complex interplay between pathway fluxes and cell function in adipocytes, and suggest a functional role for metabolic reprogramming in adipose differentiation and lipogenesis.     

Comparative study on central metabolic fluxes of <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strains in continuous culture using <Superscript>13</Superscript>C labelled substrates

Fluxes of central carbon metabolism [glycolysis, pentose phosphate pathway (PPP), tricarboxylic acid cycle (TCA cycle), biomass formation] were determined for several Bacillus megaterium strains (DSM319, WH320, WH323, MS941) in C- and N-limited chemostat cultures by ¹³C labelling experiments. The labelling patterns of proteinogenic amino acids were analysed by GC/MS and therefrom flux ratios at important nodes within the metabolic network could be calculated. On the basis of a stoichiometric metabolic model flux distributions were estimated for the different B. megaterium strains used at various cultivation conditions. Generally all strains exhibited similar metabolic flux distributions, however, several significant changes were found in (1) the glucose flux entering the PPP via the oxidative branch, (2) the reversibilities within the PPP, (3) the relative fluxes of pyruvate and acetyl-CoA fed to the TCA cycle, (4) the fluxes around the pyruvate node involving a futile cycle.