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1.
A mass flux balance-based stoichiometric model of Bacillus licheniformis for the serine alkaline protease (SAP) fermentation process has been established. The model considers 147 reaction fluxes, and there are 105 metabolites that are assumed to be in pseudo-steady state. Metabolic flux distributions were obtained from the solution of the model based on the minimum SAP accumulation rate assumption in B. licheniformis in combination with the off-line extracellular analyses of the metabolites that were the sole carbon source citrate, dry cell, organic acids, amino acids, and SAP; variations in the intracellular fluxes were demonstrated for the three periods of the batch bioprocess. The flux distribution maps showed that the cells completed the TCA cycle and utilized the gluconeogenesis pathway, pentose phosphate pathway, and anaplerotic reactions throughout the fermentation; however, the glycolysis pathway was inactive in all the periods of the fermentation. The flux values toward SAP increased throughout the bioprocess and slightly decreased in the last period; however, SAP selectivity values were almost the same in Periods II and III and higher than Period I. The diversions in the pathways and certain metabolic reactions depending on the bioprocess periods are also presented and the results indicated that the intracellular amino acid fluxes played an important role in the SAP fermentation process.  相似文献   

2.
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.  相似文献   

3.
Modelling of the fluxes in central metabolism can be performed by combining labelling experiments with metabolite balancing. Using this approach, multiple samples from a cultivation of Saccharomyces cerevisiae in metabolic and isotopic steady state were analysed, and the metabolic fluxes in central metabolism were estimated. In the various samples, the estimates of the central metabolic pathways, the tricarboxylic acid cycle, the oxidative pentose phosphate pathway and the anaplerotic pathway, showed an unprecedented reproducibility. The high reproducibility was obtained with fractional labellings of individual carbon atoms as the calculational base, illustrating that the more complex modelling using isotopomers is not necessarily superior with respect to reproducibility of the flux estimates. Based on these results some general difficulties in flux estimation are discussed.  相似文献   

4.
The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.  相似文献   

5.
Using (13)C-labeled glucose fed to the facultative alkalophilic Bacillus clausii producing the alkaline serine protease Savinase, the intracellular fluxes were quantified in continuous cultivation and in batch cultivation on a minimal medium. The flux through the pentose phosphate pathway was found to increase with increasing specific growth rate but at a much lower level than previously reported for Bacillus subtilis. Two futile cycles in the pyruvate metabolism were included in the metabolic network. A substantial flux in the futile cycle involving malic enzyme was estimated, whereas only a very small or zero flux through PEP carboxykinase was estimated, indicating that the latter enzyme was not active during growth on glucose. The uptake of the amino acids in a semirich medium containing 15 of the 20 amino acids normally present in proteins was estimated using fully labeled glucose in batch cultivations. It was found that leucine, isoleucine, and phenylalanine were taken up from the medium and not synthesized de novo from glucose. In contrast, serine and threonine were completely synthesized from other metabolites and not taken up from the medium. Valine, proline, and lysine were partly taken up from the medium and partly synthesized from glucose. The metabolic network analysis was extended to include analysis of growth on the semirich medium containing amino acids, and the metabolic flux distribution on this medium was estimated and compared with growth on minimal medium.  相似文献   

6.
Hua Q  Yang C  Baba T  Mori H  Shimizu K 《Journal of bacteriology》2003,185(24):7053-7067
The responses of Escherichia coli central carbon metabolism to knockout mutations in phosphoglucose isomerase and glucose-6-phosphate (G6P) dehydrogenase genes were investigated by using glucose- and ammonia-limited chemostats. The metabolic network structures and intracellular carbon fluxes in the wild type and in the knockout mutants were characterized by using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U-(13)C]glucose labeling and two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, glycerol, and glucose. Disruption of phosphoglucose isomerase resulted in use of the pentose phosphate pathway as the primary route of glucose catabolism, while flux rerouting via the Embden-Meyerhof-Parnas pathway and the nonoxidative branch of the pentose phosphate pathway compensated for the G6P dehydrogenase deficiency. Furthermore, additional, unexpected flux responses to the knockout mutations were observed. Most prominently, the glyoxylate shunt was found to be active in phosphoglucose isomerase-deficient E. coli. The Entner-Doudoroff pathway also contributed to a minor fraction of the glucose catabolism in this mutant strain. Moreover, although knockout of G6P dehydrogenase had no significant influence on the central metabolism under glucose-limited conditions, this mutation resulted in extensive overflow metabolism and extremely low tricarboxylic acid cycle fluxes under ammonia limitation conditions.  相似文献   

7.
A program implementing a flux model of Escherichia coli metabolism was used to analyze the effects of the addition of amino acids (tryptophan, tyrosine, phenylalanine, leucine, isoleucine, valine, histidine, lysine, threonine, cysteine, methionine, arginine, proline) to minimal medium or media lacking nitrogen, carbon, or both. The overall response of the metabolic system to the addition of various amino acids to the minimal medium is similar. Glycolysis and the synthesis of pyruvate with its subsequent degradation to acetate via acetyl-CoA become more efficient, whereas the fluxes through the pentose phosphate pathway and the TCA cycle decrease. If amino acids are used as the sole source of carbon, nitrogen, or both, the changes in the flux distribution are determined mainly by the carbon limitation. The phosphoenolpyruvate to glucose-6-phosphate flux increases; the flux through the pentose phosphate path is directed towards ribulose-5-phosphate. Other changes are determined by the compounds that are the primary products of catabolism of the added amino acid.  相似文献   

8.
To evaluate the importance of reactions within the central metabolism under different flux burdens the fluxes within the pentose phosphate pathway (PPP), as well as the other reactions of the central metabolism, were intensively analyzed and quantitated. For this purpose, Corynebacterium glutamicum was grown with [1-(13)C]glucose to metabolic and isotopic steady state and the fractional enrichments in precursor metabolites (e.g., pentose 5-phosphate) were quantified. Matrix calculus was used to express these data together with metabolite mass data. The detailed analysis of the dependence of (13)C enrichments on exchange fluxes enabled the transketolase-catalyzed exchange rate (2 pentose 5-phosphate <--> sedoheptulose 7-phosphate + glyceraldehyde 3-phosphate) to be quantified as 74.3% (molar metabolite flux) at a net flux of 10.3% and the exchange rate (pentose 5-phosphate + erythrose 4-phosphate <--> fructose 6-phosphate + glyceraldehyde 3-phosphate) to be quantified as 5.6% at a net flux of 8.1%. The flux entering the tricarboxylic acid cycle was 93.3%. The same comprehensive flux analysis as performed for the nonexcreting condition was done with the identical strain that had been forced to excrete L-glutamate. Because we had already quantified the fluxes for L-lysine excretion with an isogenic strain, three directly comparable flux situations are thus available. Consequently, this comparison permits a direct cause-and-effect relationship to be specified. In response to the different flux burdens of the cell, the PPP flux decreased from a maximum of 67% to 26%, with the glycolytic flux increasing accordingly. The carbon flux through isocitrate dehydrogenase increased from 20% to 36%. The bidirectional carbon flux between pyruvate and oxaloacetate decreased from 36% to 9%. Since the cause of the three different flux states was the allelic exchange in the final L-lysine assembling pathway or the glutamate export activity, respectively, the flexible response is the effect. This shows conclusively the enormous flexibility within the central metabolism of C. glutamicum to supply precursors upon their withdrawal for the synthesis of amino acids. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 168-180, 1997.  相似文献   

9.
The flux through the oxidative pentose phosphate (PP) pathway was estimated in Bacillus clausii, Saccharomyces cerevisiae, and Penicillium chrysogenum growing in chemostats with [1-(13)C]glucose as the limiting substrate. The flux calculations were based on a simple algebraic expression that is valid irrespective of isotope rearrangements arising from reversibilities of the reactions in the PP pathway and the upper part of the Embden-Meyerhof-Parnas pathway. The algebraically calculated fluxes were validated by comparing the results with estimates obtained using a numerical method that includes the entire central carbon metabolism. Setting the glucose uptake rate to 100, the algebraic expression yielded estimates of the PP pathway flux in B. clausii, S. cerevisiae, and P. chrysogenum of 20, 42, and 75, respectively. These results are in accordance with the results from the numerical method. The information on the labeling patterns of glucose and the proteinogenic amino acids were obtained using gas chromatography / mass spectrometry, which is a very sensitive technique, and therefore only a small amount of biomass is needed for the analysis. Furthermore, the method developed in this study is fast and readily accessible, as the calculations are based on a simple algebraic expression.  相似文献   

10.
The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden.  相似文献   

11.
Two α-amylase-producing strains of Aspergillus oryzae, a wild-type strain and a recombinant containing additional copies of the α-amylase gene, were characterized with respect to enzyme activities, localization of enzymes to the mitochondria or cytosol, macromolecular composition, and metabolic fluxes through the central metabolism during glucose-limited chemostat cultivations. Citrate synthase and isocitrate dehydrogenase (NAD) activities were found only in the mitochondria, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase (NADP) activities were found only in the cytosol, and isocitrate dehydrogenase (NADP), glutamate oxaloacetate transaminase, malate dehydrogenase, and glutamate dehydrogenase (NAD) activities were found in both the mitochondria and the cytosol. The measured biomass components and ash could account for 95% (wt/wt) of the biomass. The protein and RNA contents increased linearly with increasing specific growth rate, but the carbohydrate and chitin contents decreased. A metabolic model consisting of 69 fluxes and 59 intracellular metabolites was used to calculate the metabolic fluxes through the central metabolism at several specific growth rates, with ammonia or nitrate as the nitrogen source. The flux through the pentose phosphate pathway increased with increasing specific growth rate. The fluxes through the pentose phosphate pathway were 15 to 26% higher for the recombinant strain than for the wild-type strain.  相似文献   

12.
Metabolic fluxes estimated from stable-isotope studies provide a key to understanding cell physiology and regulation of metabolism. A limitation of the classical method for metabolic flux analysis (MFA) is the requirement for isotopic steady state. To extend the scope of flux determination from stationary to nonstationary systems, we present a novel modeling strategy that combines key ideas from isotopomer spectral analysis (ISA) and stationary MFA. Isotopic transients of the precursor pool and the sampled products are described by two parameters, D and G parameters, respectively, which are incorporated into the flux model. The G value is the fraction of labeled product in the sample, and the D value is the fractional contribution of the feed for the production of labeled products. We illustrate the novel modeling strategy with a nonstationary system that closely resembles industrial production conditions, i.e. fed-batch fermentation of Escherichia coli that produces 1,3-propanediol (PDO). Metabolic fluxes and the D and G parameters were estimated by fitting labeling distributions of biomass amino acids measured by GC/MS to a model of E. coli metabolism. We obtained highly consistent fits from the data with 82 redundant measurements. Metabolic fluxes were estimated for 20 time points during course of the fermentation. As such we established, for the first time, detailed time profiles of in vivo fluxes. We found that intracellular fluxes changed significantly during the fed-batch. The intracellular flux associated with PDO pathway increased by 10%. Concurrently, we observed a decrease in the split ratio between glycolysis and pentose phosphate pathway from 70/30 to 50/50 as a function of time. The TCA cycle flux, on the other hand, remained constant throughout the fermentation. Furthermore, our flux results provided additional insight in support of the assumed genotype of the organism.  相似文献   

13.
一种中间代谢途径代谢通量的定量分析方法   总被引:2,自引:0,他引:2  
13C标记的碳源,用二维核磁共振技术(1H-13C,HMQC)测定代谢中产生的氨基酸标记模式,研究对中间代谢途径胞内代谢通量进行定量分析的方法.通过开发软件包,改进同位素分布的数学模型,提出了反应映射矩阵(RMM)等概念.由简化算法,提高程序的执行效率,建立了定量分析胞内代谢通量的平台.代谢模型涉及了糖酵解途径、磷酸戊糖途径、三羧酸循环、几种回补反应、发酵途径和氨基酸合成途径.  相似文献   

14.
To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of (1)H-detected (13)C nuclear magnetic resonance spectroscopy to follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [1-(13)C]glucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments in carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology to C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 13.7%. Due to the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase reaction. Moreover, the total net flux of the anaplerotic reactions was quantitated as 38.0%. Most importantly, we found that in vivo one component within these anaplerotic reactions is a back flux from the carbon 4 units of the tricarboxylic acid cycle to the carbon 3 units of glycolysis of 30.6%. (c) 1996 John Wiley & Sons, Inc.  相似文献   

15.
The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using 13C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and 13C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with 13C NMR for metabolic flux analysis to reduce the use of 13C-labeled substrates and to obtain more-accurate flux values.  相似文献   

16.
The actinomycete Nonomuraea sp. ATCC 39727, producer of the glycopeptide A40926 that is used as precursor for the novel antibiotic dalbavancin, has an unusual carbon metabolism. Glucose is primarily metabolized via the Entner-Doudoroff (ED) pathway, although the energetically more favorable Embden-Meyerhof-Parnas (EMP) pathway is present in this organism. Moreover, Nonomuraea utilizes a PPi-dependent phosphofructokinase, an enzyme that has been connected with anaerobic metabolism in eukaryotes and higher plants, but recently has been recognized in several actinomycetes. In order to study its primary carbon metabolism in further detail, Nonomuraea was cultivated with [1-13C] glucose as the only carbon source and the 13C-labeling patterns of proteinogenic amino acids were determined by GC-MS analysis. Through this method, the fluxes in the central carbon metabolism during balanced growth were estimated. Moreover, a shift in the label incorporation pattern was observed in connection with phosphate limitation and increased antibiotic productivity in Nonomuraea. The shift indicated an increased flux through the EMP pathway at the expense of the flux through the ED pathway, a suggestion that was supported by alterations in intracellular metabolite levels during phosphate limitation. In contrast, expression levels of genes encoding enzymes in the ED and EMP pathways were not affected by phosphate limitation.  相似文献   

17.
The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.  相似文献   

18.
Cloutier M  Perrier M  Jolicoeur M 《Phytochemistry》2007,68(16-18):2393-2404
A dynamic model for plant cell and hairy root primary metabolism is presented. The model includes nutrient uptake (Pi, sugars, nitrogen sources), the glycolysis and pentose phosphate pathways, the TCA cycle, amino acid biosynthesis, respiratory chain, biosynthesis of cell building blocks (structural hexoses, organic acids, lipids, and organic phosphated molecules). The energy shuttles (ATP, ADP) and cofactors (NAD/H, NADP/H) are also included. The model describes the kinetics of 44 biochemical reactions (fluxes) of the primary metabolism of plant cells and includes 41 biochemical species (metabolites, nutrients, biomass components). Multiple Michaelis-Menten type kinetics are used to describe biochemical reaction rates. Known regulatory phenomena on metabolic pathways are included using sigmoid switch functions. A visualization framework showing fluxes and metabolite concentrations over time is presented. The visualization of fluxes and metabolites is used to analyze simulation results from Catharanthus roseus hairy root 50 d batch cultures. The visualization of the metabolic system allows analyzing split ratios between pathways and flux time-variations. For carbon metabolism, the cells were observed to have relatively high and stable fluxes for the central carbon metabolism and low and variable fluxes for anabolic pathways. For phosphate metabolism, a very high free intracellular Pi turnover rate was observed with higher flux variations than for the carbon metabolism. Nitrogen metabolism also exhibited large flux variations. The potential uses of the model are also discussed.  相似文献   

19.
In the present work, a novel comprehensive approach of (13)C-tracer studies with labeling measurements by MALDI-TOF MS, and metabolite balancing was developed to elucidate key fluxes in the central metabolism of lysine producing Corynebacterium glutamicum during batch culture. MALDI-TOF MS methods established allow the direct quantification of labeling patterns of low molecular mass Corynebacterium products from 1 microL of diluted culture supernatant. A mathematical model of the central Corynebacterium metabolism was developed, that describes the carbon transfer through the network via matrix calculations in a generally applicable way and calculates steady state mass isotopomer distributions of the involved metabolites. The model was applied for both experimental planning of tracer experiments and parameter estimation. Metabolic fluxes were calculated from stoichiometric data and from selected mass intensity ratios of lysine, alanine, and trehalose measured by MALDI-TOF MS in tracer experiments either with 1-(13)C glucose or with mixtures of (13)C6/(12)C6 glucose. During the phase of maximum lysine production C. glutamicum ATCC 21253 exhibited high relative fluxes into the pentose phosphate pathway of 71%, a highly reversible glucose-6-phosphate isomerase, significant backfluxes from the tricarboxylic acid cycle to the pyruvate node consuming the lysine precursor oxaloacetate, 36% net flux of anaplerotic carboxylation and 63% contribution of the dehydrogenase branch in the lysine biosynthetic pathway. Due to the straightforward and simple measurements of selected labeling patterns by MALDI-TOF MS sensitively reflecting the flux parameters of interest, the presented approach has an excellent potential to extend metabolic flux analysis from single experiments with enormous experimental effort to a broadly applied technique.  相似文献   

20.
How do intracellular fluxes respond to dynamically increasing glucose limitation when the physiology changes from strong overflow metabolism near to exclusively maintenance metabolism? Here we investigate this question in a typical industrial, glucose‐limited fed‐batch cultivation with a riboflavin overproducing Bacillus subtilis strain. To resolve dynamic flux changes, a novel approach to 13C flux analysis was developed that is based on recording 13C labeling patterns in free intracellular amino acids. Fluxes are then estimated with stationary flux ratio and iterative isotopomer balancing methods, for which a decomposition of the process into quasi‐steady states and estimation of isotopic steady state 13C labeling patterns was necessary. By this approach, we achieve a temporal resolution of 30–60 min that allows us to resolve the slow metabolic transients that typically occur in such cultivations. In the late process phase we found, most prominently, almost exclusive respiratory metabolism, significantly increased pentose phosphate pathway contribution and a strongly decreased futile cycle through the PEP carboxykinase. As a consequence, higher catabolic NADPH formation occurred than was necessary to satisfy the anabolic demands, suggesting a transhydrogenase‐like mechanism to close the balance of reducing equivalents. Biotechnol. Bioeng. 2010. 105: 795–804. © 2009 Wiley Periodicals, Inc.  相似文献   

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