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1.
The applicability of gas chromatography–combustion–isotope ratio mass spectrometry (GC–C–IRMS) for the quantification of 13C enrichment of proteinogenic amino acids in metabolic tracer experiments was evaluated. Measurement of the 13C enrichment of proteinogenic amino acids from cell hydrolyzates of Corynebacterium glutamicum growing on different mixtures containing between 0.5 and 10% [1-13C]glucose shows the significance of kinetic isotope effects in metabolic flux studies at low degree of labeling. We developed a method to calculate the 13C enrichment. The approach to correct for these effects in metabolic flux studies using δ13C measurement by GC–C–IRMS uses two parallel experiments applying substrate with natural abundance and 13C-enriched tracer substrate, respectively. The fractional enrichment obtained in natural substrate is subtracted from that of the enriched one. Tracer studies with C. glutamicum resulted in a statistically identical relative fractional enrichment of 13C in proteinogenic amino acids over the whole range of applied concentrations of [1-13C]glucose. The current findings indicate a great potential of GC–C–IRMS for labeling quantification in 13C metabolic flux analysis with low labeling degree of tracer substrate directly in larger scale bioreactors.  相似文献   

2.
Metabolic flux analysis using (13)C-labeled substrates is a well-developed method for investigating cellular behavior in steady-state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of (13)C metabolic flux analysis is proposed. An isotopomer balancing model was developed to elucidate flux distributions in the central metabolism and all amino acids synthetic pathways. A lysine-producing strain of Escherichia coli was cultivated by fed-batch mode in a growth medium containing yeast extract. Mass distribution data was derived from both intracellular free amino acids and proteinogenic amino acids measured by LC-MS/MS, and a correction parameter for the protein turnover effect on the mass distributions of intracellular amino acids was introduced. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift was detected in the fed-batch culture. The approach presented here has great potential for investigating cellular behavior in industrial processes, independent of cultivation modes, metabolic phase and growth medium.  相似文献   

3.
基于13C标记的代谢通量分析(13CMFA)由于具有准确性和应用性已成为国际上代谢工程研究的热点,其中一个至关重要的问题就是分析菌体蛋白氨基酸的13C标记丰度信息。用含20%全标记葡萄糖([U-13C])和80%天然葡萄糖的合成培养基喂养维生素B12生产菌Pseudomonas denitrifican,然后在稳态条件下取20mg带13C标记菌体用1ml 6mol/L盐酸95℃水解24h得到带13C标记的菌体蛋白氨基酸;氨基酸经分离、浓缩、真空干燥、MBDSTFA衍生化后得到的TBDMS衍生物可以用气相色谱-质谱(GC-MS)进行分析,最后分析质谱图得到15种菌体蛋白氨基酸的13C标记丰度信息。成功获得菌体蛋白氨基酸13C标记丰度信息的实验方法和样品处理技术,为13CMFA在国内进一步发展提供了参考意义。  相似文献   

4.
The novel concept of isotopic dynamic 13C metabolic flux analysis (ID-13C MFA) enables integrated analysis of isotopomer data from isotopic transient and/or isotopic stationary phase of a 13C labeling experiment, short-time experiments, and an extended range of applications of 13C MFA. In the presented work, an experimental and computational framework consisting of short-time 13C labeling, an integrated rapid sampling procedure, a LC-MS analytical method, numerical integration of the system of isotopomer differential equations, and estimation of metabolic fluxes was developed and applied to determine intracellular fluxes in glycolysis, pentose phosphate pathway (PPP), and citric acid cycle (TCA) in Escherichia coli grown in aerobic, glucose-limited chemostat culture at a dilution rate of D = 0.10 h(-1). Intracellular steady state concentrations were quantified for 12 metabolic intermediates. A total of 90 LC-MS mass isotopomers were quantified at sampling times t = 0, 91, 226, 346, 589 s and at isotopic stationary conditions. Isotopic stationarity was reached within 10 min in glycolytic and PPP metabolites. Consistent flux solutions were obtained by ID-13C MFA using isotopic dynamic and isotopic stationary 13C labeling data and by isotopic stationary 13C MFA (IS-13C MFA) using solely isotopic stationary data. It is demonstrated that integration of dynamic 13C labeling data increases the sensitivity of flux estimation, particularly at the glucose-6-phosphate branch point. The identified split ratio between glycolysis and PPP was 55%:44%. These results were confirmed by IS-13C MFA additionally using labeling data in proteinogenic amino acids (GC-MS) obtained after 5 h from sampled biomass.  相似文献   

5.
姚瑞莲 《生物工程学报》2021,37(5):1510-1525
13C代谢流量分析(13C metabolic flux analysis,13C-MFA),是通过标记实验分析蛋白氨基酸或胞内代谢物同位素标记异构体的分布情况,从而准确定量胞内反应速率.该技术在系统理解细胞代谢特性、指导代谢工程改造和揭示病理生理学等方面起着重要作用,引起研究者的广泛重视.文中重点综述了代谢流分析30...  相似文献   

6.
Sekiyama Y  Kikuchi J 《Phytochemistry》2007,68(16-18):2320-2329
Novel technologies for measuring biological systems and methods for visualizing data have led to a revolution in the life sciences. Nuclear magnetic resonance (NMR) techniques can provide information on metabolite structure and metabolic dynamics at the atomic level. We have been developing a new method for measuring the dynamic metabolic network of crude extracts that combines [(13)C(6)]glucose stable isotope labeling of Arabidopsis thaliana and multi-dimensional heteronuclear NMR analysis, whereas most conventional metabolic flux analyses examine proteinogenic amino acids that are specifically labeled with partially labeled substrates such as [2-(13)C(1)]glucose or 10% [(13)C(6)]glucose. To show the validity of our method, we investigated how to obtain information about biochemical reactions, C-C bond formation, and the cleavage of the main metabolites, such as free amino acids, in crude extracts based on the analysis of the (13)C-(13)C coupling pattern in 2D-NMR spectra. For example, the combination of different extraction solvents allows one to distinguish complicated (13)C-(13)C fine couplings at the C2 position of amino acids. As another approach, f1-f3 projection of the HCACO spectrum also helps in the analysis of (13)C-(13)C connectivities. Using these new methods, we present an example that involves monitoring the incorporation profile of [(13)C(6)]glucose into A. thaliana and its metabolic dynamics, which change in a time-dependent manner with atmospheric (12)CO(2) assimilation.  相似文献   

7.
We have developed a novel approach for measuring highly accurate and precise metabolic fluxes in living cells, termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. The COMPLETE-MFA method is based on combined analysis of multiple isotopic labeling experiments, where the synergy of using complementary tracers greatly improves the precision of estimated fluxes. In this work, we demonstrate the COMPLETE-MFA approach using all singly labeled glucose tracers, [1-13C], [2-13C], [3-13C], [4-13C], [5-13C], and [6-13C]glucose to determine precise metabolic fluxes for wild-type Escherichia coli. Cells were grown in six parallel cultures on defined medium with glucose as the only carbon source. Mass isotopomers of biomass amino acids were measured by gas chromatography–mass spectrometry (GC–MS). The data from all six experiments were then fitted simultaneously to a single flux model to determine accurate intracellular fluxes. We obtained a statistically acceptable fit with more than 300 redundant measurements. The estimated flux map is the most precise flux result obtained thus far for E. coli cells. To our knowledge, this is the first time that six isotopic labeling experiments have been successfully integrated for high-resolution 13C-flux analysis.  相似文献   

8.
9.
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   

10.
Biosynthetically directed fractional 13C labeling of the proteinogenic amino acids is achieved by feeding a mixture of uniformly 13C-labeled and unlabeled carbon source compounds into a bioreaction network. Analysis of the resulting labeling pattern enables both a comprehensive characterization of the network topology and the determination of metabolic flux ratios. Attractive features with regard to routine applications are (i) an inherently small demand for 13C-labeled source compounds and (ii) the high sensitivity of two-dimensional [13C,1H]-correlation nuclear magnetic resonance spectroscopy for analysis of 13C-labeling patterns. A user-friendly program, FCAL, is available to allow rapid data analysis. This novel approach, which recently also has been employed in conjunction with metabolic flux balancing to obtain reliable estimates of in vivo fluxes, enables efficient support of metabolic engineering and biotechnology process design.  相似文献   

11.
This study focuses on unravelling the carbon and redox metabolism of a previously developed glycerol-overproducing Saccharomyces cerevisiae strain with deletions in the structural genes encoding triosephosphate isomerase (TPI1), the external mitochondrial NADH dehydrogenases (NDE1 and NDE2) and the respiratory chain-linked glycerol-3-phosphate dehydrogenase (GUT2). Two methods were used for analysis of metabolic fluxes: metabolite balancing and (13)C-labelling-based metabolic flux analysis. The isotopic enrichment of intracellular primary metabolites was measured both directly (liquid chromatography-MS) and indirectly through proteinogenic amino acids (nuclear magnetic resonance and gas chromatography-MS). Because flux sensitivity around several important metabolic nodes proved to be dependent on the applied technique, the combination of the three (13)C quantification techniques generated the most accurate overall flux pattern. When combined, the measured conversion rates and (13)C-labelling data provided evidence that a combination of assimilatory metabolism and pentose phosphate pathway activity diverted some of the carbon away from glycerol formation. Metabolite balancing indicated that this results in excess cytosolic NADH, suggesting the presence of a cytosolic NADH sink in addition to those that were deleted. The exchange flux of four-carbon dicarboxylic acids across the mitochondrial membrane, as measured by the (13)C-labelling data, supports a possible role of a malate/aspartate or malate/oxaloacetate redox shuttle in the transfer of these redox equivalents from the cytosol to the mitochondrial matrix.  相似文献   

12.
Information on metabolic networks could provide the basis for the design of targets for metabolic engineering. To study metabolic flux in cereals, developing maize (Zea mays) kernels were grown in sterile culture on medium containing [U-(13)C(6)]glucose or [1,2-(13)C(2)]acetate. After growth, amino acids, lipids, and sitosterol were isolated from kernels as well as from the cobs, and their (13)C isotopomer compositions were determined by quantitative nuclear magnetic resonance spectroscopy. The highly specific labeling patterns were used to analyze the metabolic pathways leading to amino acids and the triterpene on a quantitative basis. The data show that serine is generated from phosphoglycerate, as well as from glycine. Lysine is formed entirely via the diaminopimelate pathway and sitosterol is synthesized entirely via the mevalonate route. The labeling data of amino acids and sitosterol were used to reconstruct the labeling patterns of key metabolic intermediates (e.g. acetyl-coenzyme A, pyruvate, phosphoenolpyruvate, erythrose 4-phosphate, and Rib 5-phosphate) that revealed quantitative information about carbon flux in the intermediary metabolism of developing maize kernels. Exogenous acetate served as an efficient precursor of sitosterol, as well as of amino acids of the aspartate and glutamate family; in comparison, metabolites formed in the plastidic compartments showed low acetate incorporation.  相似文献   

13.
A novel method for metabolic flux studies of central metabolism which is based on respirometric (13)C flux analysis, i.e., parallel (13)C tracer studies with online CO(2) labeling measurements is applied to flux quantification of a lysine-producing mutant of Corynebacterium glutamicum. For this purpose, 3 respirometric (13)C labeling experiments with [1-(13)C(1)], [6-(13)C(1)] and [1,6-(13)C(2)] glucose were carried out in parallel. All fluxes comprising the reactions of glycolysis, of TCA cycle, of C3- and C4-metabolite interconversion and of lysine biosynthesis as well as the net reactions in the pentose phosphate pathway could be quantified solely using experimental data obtained from CO(2) labeling and extracellular rate measurements. At key branch points, 68+/-5% of glucose 6-phosphate were observed to be metabolized into pentose phosphate pathway and 48+/-1% of pyruvate into TCA cycle via pyruvate dehydrogenase. The results showed a good agreement with the previous studies using (13)C tracer cultivation and GC/MS analysis of proteinogenic amino acids. Also, respiratory quotient calculated from flux estimates using redox balance showed a high accordance with the value determined directly from the measured specific rates of O(2) consumption and CO(2) production. The results strongly support that the respirometric (13)C metabolic flux analysis is suited as an alternative to the conventional methods to study functional and regulatory activities of cells. The developed method is applicable to study growing or non-growing cells, primary and secondary metabolism and immobilized cells. Due to the non-accumulating nature of CO(2) labeling and instantaneous nature of the resulting fluxes, the method can also be used for dynamic profiling of metabolic activities. Therefore, it is complementary to conventional methods for metabolic flux analysis.  相似文献   

14.
We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteinogenic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1-13C] and [U-13C]glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently, the low in vivo activity of the Entner-Doudoroff pathway in wild-type E. coli increased up to a contribution of 30% to glucose catabolism in mutants of glycolysis and TCA cycle. Moreover, glucose 6-phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis, suggesting a yet unidentified bypass of this reaction. Although strongly affected by environmental conditions, a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism. Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption.  相似文献   

15.
Microbial production of many commercially important secondary metabolites occurs during stationary phase, and methods to measure metabolic flux during this growth phase would be valuable. Metabolic flux analysis is often based on isotopomer information from proteinogenic amino acids. As such, flux analysis primarily reflects the metabolism pertinent to the growth phase during which most proteins are synthesized. To investigate central metabolism and amino acids synthesis activity during stationary phase, addition of fully 13C‐labeled glucose followed by induction of green fluorescent protein (GFP) expression during stationary phase was used. Our results indicate that Escherichia coli was able to produce new proteins (i.e., GFP) in the stationary phase, and the amino acids in GFP were mostly from degraded proteins synthesized during the exponential growth phase. Among amino acid biosynthetic pathways, only those for serine, alanine, glutamate/glutamine, and aspartate/asparagine had significant activity during the stationary phase. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010  相似文献   

16.
Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.  相似文献   

17.
Metabolic flux analysis is a powerful diagnostic tool in metabolic engineering, and determination of biomass composition is indispensable to accurate flux evaluation. We report the elemental and biomolecular composition of Catharanthus roseus hairy roots, a pharmaceutically significant plant system and an important metabolic engineering target. The molecular formula of the organic material in the hairy roots was C12.0H22.7N0.4O7.6 during mid-exponential growth. The abundances of lipids, lignin, cellulose, hemicellulose, starch, protein, proteinogenic amino acids, mineral ash, and moisture in the biomass were quantified. Analysis of water-soluble components of the biomass with 1-D 13C and 2-D [1H,1H] correlation (COSY) NMR spectroscopy revealed that the water-soluble components were composed almost entirely of -glucans. Agropine, a frequently reported hairy root biomass component, was not detected. Our measurements of the biomass components quantified 83.6 +/- 9.3% (w/w) of the biomass. Together with previously reported abundances of indole alkaloids, we accounted for at least 85.9 +/- 11.6% (w/w) of the carbon in the biomass, which enabled the precise determination of 12 biomass synthesis fluxes.  相似文献   

18.
Uniformly (13)C labeled glucose was fed to a lactic acid bacterium growing on a defined medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with (13)C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange flux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.  相似文献   

19.
Attaining metabolic and isotopic balanced growth is one critical condition for physiological studies using isotope-labeled tracers, but is very difficult to obtain in batch culture due to the extensive metabolite exchange with the surrounding medium and related physiological changes. In the present study, we investigated metabolic and isotopic behavior of CHO cells in differently designed media. We observed that the assumption of balanced cell growth cannot be justified in batch culture of CHO cells directly using conventional, commercially available media. By systematically redesigning media composition and characterizing metabolic steady state based on mass balances and measurement of labeling dynamics, we achieved balanced cell growth for the main cellular substrates in CHO cells. This was done in a step-by-step analysis of growth and primary metabolism of CHO cells with the use of [U-13C]glucose feeding and adjusting concentrations of amino acids in the growth medium. The optimized media obtained at the end of the study provide balanced growth and isotopic steady state or at least asymptotic steady state. As a result, we established a platform to conduct isotope-based physiological studies of mammalian systems more reliably and therefore well suited for later use in metabolic profiling of mammalian systems such as 13C-labeled metabolic flux analysis.  相似文献   

20.
Rigorous mathematical modeling of carbon-labeling experiments allows estimation of fluxes through the pathways of central carbon metabolism, yielding powerful information for basic scientific studies as well as for a wide range of applications. However, the mathematical models that have been developed for flux determination from 13C labeling data have commonly neglected the influence of kinetic isotope effects on the distribution of 13C label in intracellular metabolites, as these effects have often been assumed to be inconsequential. We have used measurements of the 13C isotope effects on the pyruvate dehydrogenase enzyme from the literature to model isotopic fractionation at the pyruvate node and quantify the modeling errors expected to result from the assumption that isotope effects are negligible. We show that under some conditions kinetic isotope effects have a significant impact on the 13C labeling patterns of intracellular metabolites, and the errors associated with neglecting isotope effects in 13C-metabolic flux analysis models can be comparable in size to measurement errors associated with GC–MS. Thus, kinetic isotope effects must be considered in any rigorous assessment of errors in 13C labeling data, goodness-of-fit between model and data, confidence intervals of estimated metabolic fluxes, and statistical significance of differences between estimated metabolic flux distributions.  相似文献   

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