76 research outputs found
Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum
BACKGROUND: Quantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates. RESULTS: Quantification of the fluxes of the elementary modes in the metabolism of C. glutamicum was formulated as linear programming. The analysis demonstrated that the solution was dependent on the criteria of objective function when less than four accumulation rates of the external metabolites were considered. The analysis yielded feasible ranges of fluxes of elementary modes that satisfy the experimental accumulation rates. In C. glutamicum, the elementary modes relating to biomass synthesis through glycolysis and TCA cycle were predominantly operational in the initial growth phase. At a later time, the elementary modes contributing to lysine synthesis became active. The oxygen and ammonia uptake rates were shown to be bounded in the phenotypic space due to the stoichiometric constraint of the elementary modes. CONCLUSION: We have demonstrated the use of elementary modes and the linear programming to quantify a metabolic network. We have used the methodology to quantify the network of C. glutamicum, which evaluates the set of operational elementary modes at different phases of fermentation. The methodology was also used to determine the feasible solution space for a given set of substrate uptake rates under specific optimization criteria. Such an approach can be used to determine the optimality of the accumulation rates of any metabolite in a given network
Engineering yield and rate of reductive biotransformation in Escherichia coli by partial cyclization of the pentose phosphate pathway and PTS-independent glucose transport
Optimization of yields and productivities in reductive whole-cell biotransformations is an important issue for the industrial application of such processes. In a recent study with Escherichia coli, we analyzed the reduction of the prochiral β-ketoester methyl acetoacetate by an R-specific alcohol dehydrogenase (ADH) to the chiral hydroxy ester (R)-methyl 3-hydroxybutyrate (MHB) using glucose as substrate for the generation of NADPH. Deletion of the phosphofructokinase gene pfkA almost doubled the yield to 4.8 mol MHB per mole of glucose, and it was assumed that this effect was due to a partial cyclization of the pentose phosphate pathway (PPP). Here, this partial cyclization was confirmed by 13C metabolic flux analysis, which revealed a negative net flux from glucose 6-phosphate to fructose 6-phosphate catalyzed by phosphoglucose isomerase. For further process optimization, the genes encoding the glucose facilitator (glf) and glucokinase (glk) of Zymomonas mobilis were overexpressed in recombinant E. coli strains carrying ADH and deletions of either pgi (phosphoglucose isomerase), or pfkA, or pfkA plus pfkB. In all cases, the glucose uptake rate was increased (30–47%), and for strains Δpgi and ΔpfkA also, the specific MHB production rate was increased by 15% and 20%, respectively. The yield of the latter two strains slightly dropped by 11% and 6%, but was still 73% and 132% higher compared to the reference strain with intact pgi and pfkA genes and expressing glf and glk. Thus, metabolic engineering strategies are presented for improving yield and rate of reductive redox biocatalysis by partial cyclization of the PPP and by increasing glucose uptake, respectively
Identification and application of a different glucose uptake system that functions as an alternative to the phosphotransferase system in Corynebacterium glutamicum
Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to uptake and phosphorylate glucose; no other route has yet been identified. Disruption of the ptsH gene in wild-type C. glutamicum resulted, as expected, in a phenotype exhibiting little growth on any of the PTS sugars: glucose, fructose, and sucrose. However, a suppressor mutant that grew on glucose but not on the other two sugars was spontaneously isolated from the PTS-negative strain WT Delta ptsH. The suppressor strain SPH2, unlike the wild-type strain, exhibited a phenotype of resistance to 2-deoxyglucose which is known to be a toxic substrate for the glucose-PTS of this microbe, suggesting that strain SPH2 utilizes glucose via a different system involving a permease and native glucokinases. Analysis of the C. glutamicum genome sequence using Escherichia coli galactose permease, which can transport glucose, led to the identification of two candidate genes, iolT1 and iolT2, both of which have been reported as myo-inositol transporters. When cultured on glucose medium supplemented with myo-inositol, strain WT Delta ptsH was able to consume glucose, suggesting that glucose uptake was mediated by one or more myo-inositol-induced transporters. Overexpression of iolT1 alone and that of iolT2 alone under the gapA promoter in strain WT Delta ptsH rendered the strain capable of growing on glucose, proving that each transporter played a role in glucose uptake. Disruption of iolT1 in strain SPH2 abolished growth on glucose, whereas disruption of iolT2 did not, revealing that iolT1 was responsible for glucose uptake in strain SPH2. Sequence analysis of the iol gene cluster and its surrounding region identified a single-base deletion in the putative transcriptional regulator gene Cgl0157 of strain SPH2. Introduction of the frameshift mutation allowed strain WT Delta ptsH to grow on glucose, and further deletion of iolT1 abolished the growth again, indicating that inactivation of Cgl0157 under a PTS-negative background can be a means by which to express the iolT1-specified glucose uptake bypass instead of the native PTS. When this strategy was applied to a defined lysine producer, the engineered strain displayed increased lysine production from glucose.ArticleAPPLIED MICROBIOLOGY AND BIOTECHNOLOGY. 90(4):1443-1451 (2011)journal articl
13C Metabolic Flux Analysis Identifies an Unusual Route for Pyruvate Dissimilation in Mycobacteria which Requires Isocitrate Lyase and Carbon Dioxide Fixation
Mycobacterium tuberculosis requires the enzyme isocitrate lyase (ICL) for growth and virulence in vivo. The demonstration that M. tuberculosis also requires ICL for survival during nutrient starvation and has a role during steady state growth in a glycerol limited chemostat indicates a function for this enzyme which extends beyond fat metabolism. As isocitrate lyase is a potential drug target elucidating the role of this enzyme is of importance; however, the role of isocitrate lyase has never been investigated at the level of in vivo fluxes. Here we show that deletion of one of the two icl genes impairs the replication of Mycobacterium bovis BCG at slow growth rate in a carbon limited chemostat. In order to further understand the role of isocitrate lyase in the central metabolism of mycobacteria the effect of growth rate on the in vivo fluxes was studied for the first time using 13C-metabolic flux analysis (MFA). Tracer experiments were performed with steady state chemostat cultures of BCG or M. tuberculosis supplied with 13C labeled glycerol or sodium bicarbonate. Through measurements of the 13C isotopomer labeling patterns in protein-derived amino acids and enzymatic activity assays we have identified the activity of a novel pathway for pyruvate dissimilation. We named this the GAS pathway because it utilizes the Glyoxylate shunt and Anapleurotic reactions for oxidation of pyruvate, and Succinyl CoA synthetase for the generation of succinyl CoA combined with a very low flux through the succinate – oxaloacetate segment of the tricarboxylic acid cycle. We confirm that M. tuberculosis can fix carbon from CO2 into biomass. As the human host is abundant in CO2 this finding requires further investigation in vivo as CO2 fixation may provide a point of vulnerability that could be targeted with novel drugs. This study also provides a platform for further studies into the metabolism of M. tuberculosis using 13C-MFA
Anaplerotische Reaktionen in : Untersuchungen zur Bedeutung der PEP-Carboxylase und der Pyruvat-Carboxylase im Zentralstoffwechsel und bei der Aminosäure-Produktion
Anaplerotische Reaktionen dienen dazu, während des Wachstums und der Aminosäure-Produktion den Tricarbonsäure-Zyklus kontinuierlich mit Oxalacetat aufzufüllen. Diese Funktion wird bei Wachstum auf Kohlenhydraten durch Carboxylierung von PEP oder Pyruvat zu Oxalacetat übernommen. Das Ziel der vorliegenden Arbeit war die Charakterisierung anaplerotischer C-carboxylierender Enzyme in und die Aufklärung der Rolle dieser Enzyme für das Wachstum und die Aminosäure-Produktion. 1. Eine definierte PEP-Carboxylase-negative Mutante von zeigte auf Minimalmedium mit Glucose das gleiche Wachstum wie der Wildtyp, und eine entsprechende Mutante des Lysin-Produktionsstamms MH20-22B produzierte gleich viel Lysin wie der Ausgangsstamm. Das Ergebnis zeigt, daß die PEP-Carboxylase für das Wachstum und die Aminosäure-Produktion von nicht essentiell ist. Damit war nachgewiesen, daß über eine weitere anaplerotische Sequenz verfügt, die die PEP-Carboxylase ersetzen kann. 2. Durch die physiologische Charakterisierung definierter PEP-Carboxylase- / Isocitrat-Lyase-Doppelmutanten konnte der Glyoxylat-Zyklus als alternative anaplerotische Sequenz bei Wachstum auf Glucose oder Lactat ausgeschlossen werden. HCO-Markierungsexperimente ergaben, daß die PEP-Carboxylase-negative Mutante wie der Wildtyp eine Carboxylierung von PEP oder Pyruvat katalysiert. Das zeigt, daß das zur PEP-Carboxylase alternative Enzym eine C-Carboxylierung katalysiert. 3. In konnte eine IDP/GDP-abhängige PEP-Carboxykinase nachgewiesen werden, die sowohl die Carboxylierung von PEP zu Oxalacetat mit einer spezifischen Aktivität von ca. 80 mU/mg Protein als auch die Decarboxylierung von Oxalacetat zu PEP mit einer spezifischen Aktivität von ca. 1 U/mg Protein katalysiert. Die 10-fach niedrigere carboxylierende Aktivität und die Hemmung der PEP-Carboxykinase in der anaplerotischen Richtung durch ATP und ADP deuten an, daß die PEP-Carboxykinase eine gluconeogenetische Funktion hat. Neben der PEP-Carboxykinase konnten in auch das Malat-Enzym und die Oxalacetat-Decarboxylase nachgewiesen und als anaplerotisch aktive Enzyme ausgeschlossen werden. 4. In permeabilisierten Zellen von konnte die Pyruvat-Carboxylase mit spezifischen Aktivitäten von ca. 15 mU/mg TG bei Wachstum auf Glucose und ca. 50 mU/mg TG bei Wachstum auf Lactat nachgewiesen werden. Das Enzym wird sowohl durch ADP (K = 2,6 mM) und AMP (K = 0,76 mM) als auch durch Acetyl-CoA (K = 0,11 mM) inhibiert. Mit der Pyruvat-Carboxylase verfügt über ein zur PEP-Carboxylase alternatives anaplerotisches Enzym. [...
Role of 3-phosphoglycerate mutase and pyruvate kinase in Corynebacterium glutamicum for growth and serine production
A concept for sustainable biotechnological amino acid production: The example L-serine. Bioperspectives zus.
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