Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum

This study addresses the relation between NADPH supply and penicillin synthesis, by comparing the flux through the oxidative branch of the pentose phosphate pathway (PPP; the main source of cytosolic NADPH) in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. The fluxes through the oxidative part of the PPP were determined using the recently introduced gluconate-tracer method. Significantly higher oxidative PPP fluxes were observed in penicillin-G producing chemostat cultures, indicating that penicillin production puts a major burden on the supply of cytosolic NADPH. To our knowledge this is the first time direct experimental proof is presented for the causal relationship between penicillin production and NADPH supply. Additional insight in the metabolism of P. chrysogenum was obtained by comparing the PPP fluxes from the gluconate-tracer experiment to oxidative PPP fluxes derived via metabolic flux analysis, using different assumptions for the stoichiometry of NADPH consumption and production.     

A metabolome study of the steady-state relation between central metabolism, amino acid biosynthesis and penicillin production in Penicillium chrysogenum

The relation between central metabolism and the penicillin biosynthesis pathway in Penicillium chrysogenum was studied by manipulating the steady-state flux in both pathways. A high producing industrial strain was cultivated at a growth rate mu=0.05 h(-1) in glucose-limited chemostat cultures, both under penicillin-G producing and non-producing conditions. Non-producing conditions were accomplished in two ways: (1) by cultivation without addition of the side chain precursor phenylacetic acid and (2) by cultivation of a mutant strain which lost all copies of the gene cluster coding for the penicillin biosynthesis pathway. Manipulation of the fluxes through central metabolism was obtained by cultivation on either glucose or ethanol as sole carbon source. A positive relation was observed between metabolite concentrations and carbon flux in central metabolism. Furthermore, in many cases a positive relation was found between the concentrations of free amino acids and their direct precursors in central metabolism. This corresponds with control of the biosynthesis of these amino acids via feed back inhibition by the end product. With respect to the penicillin production pathway, the flux seems not influenced by two of the three precursor amino acids, namely alphaAAA and valine but is only influenced by cysteine, which requires a large NADPH supply, and the ATP level. An interesting observation is that the absence of penicillin production seems to stimulate storage metabolism (trehalose metabolism). This leads to the final conclusion that the penicillin production flux appears to be mostly influenced by the availability of energy and redox cofactors, where ATP is supposed to exert its influence at ACV-synthetase and NADPH at the cysteine level.     

Metabolic adaptation of Escherichia coli during temperature-induced recombinant protein production: 2. Redirection of metabolic fluxes

The impact of temperature-induced synthesis of human basic fibroblast growth factor (hFGF-2) in high-cell-density cultures of recombinant Escherichia coli was studied by estimating metabolic flux variations. Metabolic flux distributions in E. coli were calculated by means of a stoichiometric network and linear programming. After the temperature upshift, a substantially elevated energy demand for synthesis of hFGF-2 and heat shock proteins resulted in a redirection of metabolic fluxes. Catabolic pathways like the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid (TCA) cycle showed significantly enhanced activities, leading to reduced flux to growth-associated pathways like the pentose phosphate pathway and other anabolic pathways. Upon temperature upshift, an excess of NADPH was produced in the TCA cycle by isocitrate dehydrogenase. The metabolic model predicted the involvement of a transhydrogenase generating additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. The influence of the temperature upshift on the host's metabolism was investigated by means of a control strain harboring the "empty" parental expression vector. The metabolic fluxes after the temperature upshift were redirected similarly to the production strain; the effects, however, were observed to a lesser extent and with different time profiles.     

Understanding fatty acid synthesis in developing maize embryos using metabolic flux analysis

The efficiency with which developing maize embryos convert substrates into seed storage reserves was determined to be 57–71%, by incubating developing maize embryos with uniformly labeled ¹⁴C substrates and measuring their conversion to CO₂ and biomass products. To map the pattern of metabolic fluxes underlying this efficiency, maize embryos were labeled to isotopic steady state using a combination of labeled ¹³C-substrates. Intermediary metabolic fluxes were estimated by computer-aided modeling of the central metabolic network using the labeling data collected by NMR and GC-MS and the biomass composition. The resultant flux map reveals that even though 36% of the entering carbon goes through the oxidative pentose-phosphate pathway, this does not fully meet the NADPH demands for fatty acid synthesis. Metabolic flux analysis and enzyme activities highlight the importance of plastidic NADP-dependent malic enzyme, which provides one-third of the carbon and NADPH required for fatty acid synthesis in developing maize embryos.     

Isotopically nonstationary 13C metabolic flux analysis in resting and activated human platelets

Platelet metabolism is linked to platelet hyper- and hypoactivity in numerous human diseases. Developing a detailed understanding of the link between metabolic shifts and platelet activation state is integral to improving human health. Here, we show the first application of isotopically nonstationary ¹³C metabolic flux analysis to quantitatively measure carbon fluxes in both resting and thrombin activated platelets. Metabolic flux analysis results show that resting platelets primarily metabolize glucose to lactate via glycolysis, while acetate is oxidized to fuel the tricarboxylic acid cycle. Upon activation with thrombin, a potent platelet agonist, platelets increase their uptake of glucose 3-fold. This results in an absolute increase in flux throughout central metabolism, but when compared to resting platelets they redistribute carbon dramatically. Activated platelets decrease relative flux to the oxidative pentose phosphate pathway and TCA cycle from glucose and increase relative flux to lactate. These results provide the first report of reaction-level carbon fluxes in platelets and allow us to distinguish metabolic fluxes with much higher resolution than previous studies.     

13C-Labeled metabolic flux analysis of a fed-batch culture of elutriated Saccharomyces cerevisiae

This study addresses the question of whether observable changes in fluxes in the primary carbon metabolism of Saccharomyces cerevisiae occur between the different phases of the cell division cycle. To detect such changes by metabolic flux analysis, a 13C-labeling experiment was performed with a fed-batch culture inoculated with a partially synchronized cell population obtained through centrifugal elutriation. Such a culture exhibits dynamic changes in the fractions of cells in different cell cycle phases over time. The mass isotopomer distributions of free intracellular metabolites in central carbon metabolism were measured by liquid chromatography-mass spectrometry. For four time points during the culture, these distributions were used to obtain the best estimates for the metabolic fluxes. The obtained flux fits suggested that the optimally fitted split ratio for the pentose phosphate pathway changed by almost a factor of 2 up and down around a value of 0.27 during the experiment. Statistical analysis revealed that some of the fitted flux distributions for different time points were significantly different from each other, indicating that cell cycle-dependent variations in cytosolic metabolic fluxes indeed occurred.     

从代谢流量分析角度探讨培养条件改变下对放射型根瘤茵WSH2601合成辅酶Q10的影响

分析了放射型根瘤菌(R．radiobacter)WSH2601生物合成辅酶Q10的代谢途径网络,并在溶氧条件改变和培养基中添加玉米浆条件下对辅酶Q10发酵细胞内代谢途径流量变化作定量的分析,结果表明：提高溶氧浓度(20％)5-磷酸核酮糖(RuSP)物流(r7)增加26．6,即糖酵解途径(EMP)途径向磷酸戊糖途径(HMP)转移;添加1％玉米浆r7增加17．2,EMP与HMP途径物流比值与三羧酸循环(TCA)途径物流都下降,而癸异戊烯基焦磷酸(DPP)生成物流通量(绝对值)变化都较小,即辅酶Q10的生物合成更大程度地取决于辅酶Q10生物合成途径中催化DPP的合成和4-羟基苯甲酸(PHB)与DPP的缩合反应的两种关键酶活性。6-磷酸葡萄糖(G6P)节点是辅酶Q10生物合成代谢途径的柔性节点,而丙酮酸节点是半柔性节点。细胞生物量的提高与HMP途径物流增加有关。     

Analysis of metabolic flux in <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing human-like collagen in fed-batch culture

Metabolic flux distributions of recombinant Escherichia coli BL21 expressing human-like collagen were determined by means of a stoichiometric network and metabolic balancing. At the batch growth stage, the fluxes of the pentose phosphate pathway were higher than the fluxes of the fed-batch growth phase and the production stage. After the temperature was increased, there was a substantially elevated energy demand for synthesizing human-like collagen and heat-shock proteins, which resulted in changes in metabolic fluxes. The activities of the Embden-Meyerhof-Parnas pathway and the tricarboxylic acid cycle were significantly enhanced, leading to a reduction in the fluxes of the pentose phosphate pathway and other anabolic pathways. The temperature upshift also caused an increase in NADPH production by isocitrate dehydrogenase in the tricarboxylic acid cycle. The metabolic model predicted the involvement of a transhydrogenase that generates additional NADH from NADPH, thereby increasing ATP regeneration in the respiratory chain. These data indicated that the maintenance energy for cellular activity increased with the increase in biomass in fed-batch culture, and that cell growth and synthesis of human-like collagen could clearly represent the changes in metabolic fluxes. At the production stage, more NADPH was used to synthesize human-like collagen than for maintaining cellular activity, cell growth, and cell propagation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Improved oxytetracycline production in Streptomyces rimosus M4018 by metabolic engineering of the G6PDH gene in the pentose phosphate pathway

The aromatic polyketide antibiotic, oxytetracycline (OTC), is produced by Streptomyces rimosus as an important secondary metabolite. High level production of antibiotics in Streptomycetes requires precursors and cofactors which are derived from primary metabolism; therefore it is exigent to engineer the primary metabolism. This has been demonstrated by targeting a key enzyme in the oxidative pentose phosphate pathway (PPP) and nicotinamide adenine dinucleotide phosphate (NADPH) generation, glucose-6-phosphate dehydrogenase (G6PDH), which is encoded by zwf1 and zwf2. Disruption of zwf1 or zwf2 resulted in a higher production of OTC. The disrupted strain had an increased carbon flux through glycolysis and a decreased carbon flux through PPP, as measured by the enzyme activities of G6PDH and phosphoglucose isomerase (PGI), and by the levels of ATP, which establishes G6PDH as a key player in determining carbon flux distribution. The increased production of OTC appeared to be largely due to the generation of more malonyl-CoA, one of the OTC precursors, as observed in the disrupted mutants. We have studied the effect of zwf modification on metabolite levels, gene expression, and secondary metabolite production to gain greater insight into flux distribution and the link between the fluxes in the primary and secondary metabolisms.     

Metabolic flux analysis of Escherichia coli K12 grown on 13C-labeled acetate and glucose using GC-MS and powerful flux calculation method

A new algorithm was developed for the estimation of the metabolic flux distribution based on GC-MS data of proteinogenic amino acids. By using a sensitive GC-MS protocol as well as by combining the global search algorithm such as the genetic algorithm with the local search algorithm such as the Levenberg-Marquardt algorithm, not only the distribution of the net fluxes in the entire network, but also certain exchange fluxes which contribute significantly to the isotopomer distribution could be quantified. This mass isotopomer analysis could identify the biochemical changes involved in the regulation where acetate or glucose was used as a main carbon source. The metabolic flux analysis clearly revealed that when the specific growth rate increased, only a slight change in flux distribution was observed for acetate metabolism, indicating that subtle regulation mechanism exists in certain key junctions of this network system. Different from acetate metabolism, when glucose was used as a carbon source, as the growth rate increased, a significant increase in relative pentose phosphate pathway (PPP) flux was observed for Escherichia coli K12 at the expense of the citric acid cycle, suggesting that when growing on glucose, the flux catalyzed by isocitrate dehydrogenase could not fully fulfill the NADPH demand for cell growth, causing the oxidative PPP to be utilized to a larger extent so as to complement the NADPH demand. The GC-MS protocol as well as the new algorithm demonstrated here proved to be a powerful tool for characterizing metabolic regulation and can be utilized for strain improvement and bioprocess optimization.     

Quantification of compartmented metabolic fluxes in developing soybean embryos by employing biosynthetically directed fractional (13)C labeling, two-dimensional [(13)C, (1)H] nuclear magnetic resonance, and comprehensive isotopomer balancing

Metabolic flux quantification in plants is instrumental in the detailed understanding of metabolism but is difficult to perform on a systemic level. Toward this aim, we report the development and application of a computer-aided metabolic flux analysis tool that enables the concurrent evaluation of fluxes in several primary metabolic pathways. Labeling experiments were performed by feeding a mixture of U-(13)C Suc, naturally abundant Suc, and Gln to developing soybean (Glycine max) embryos. Two-dimensional [(13)C, (1)H] NMR spectra of seed storage protein and starch hydrolysates were acquired and yielded a labeling data set consisting of 155 (13)C isotopomer abundances. We developed a computer program to automatically calculate fluxes from this data. This program accepts a user-defined metabolic network model and incorporates recent mathematical advances toward accurate and efficient flux evaluation. Fluxes were calculated and statistical analysis was performed to obtain sds. A high flux was found through the oxidative pentose phosphate pathway (19.99 +/- 4.39 micromol d(-1) cotyledon(-1), or 104.2 carbon mol +/- 23.0 carbon mol per 100 carbon mol of Suc uptake). Separate transketolase and transaldolase fluxes could be distinguished in the plastid and the cytosol, and those in the plastid were found to be at least 6-fold higher. The backflux from triose to hexose phosphate was also found to be substantial in the plastid (21.72 +/- 5.00 micromol d(-1) cotyledon(-1), or 113.2 carbon mol +/-26.0 carbon mol per 100 carbon mol of Suc uptake). Forward and backward directions of anaplerotic fluxes could be distinguished. The glyoxylate shunt flux was found to be negligible. Such a generic flux analysis tool can serve as a quantitative tool for metabolic studies and phenotype comparisons and can be extended to other plant systems.     

13C-labeled gluconate tracing as a direct and accurate method for determining the pentose phosphate pathway split ratio in Penicillium chrysogenum

In this study we developed a new method for accurately determining the pentose phosphate pathway (PPP) split ratio, an important metabolic parameter in the primary metabolism of a cell. This method is based on simultaneous feeding of unlabeled glucose and trace amounts of [U-13C]gluconate, followed by measurement of the mass isotopomers of the intracellular metabolites surrounding the 6-phosphogluconate node. The gluconate tracer method was used with a penicillin G-producing chemostat culture of the filamentous fungus Penicillium chrysogenum. For comparison, a 13C-labeling-based metabolic flux analysis (MFA) was performed for glycolysis and the PPP of P. chrysogenum. For the first time mass isotopomer measurements of 13C-labeled primary metabolites are reported for P. chrysogenum and used for a 13C-based MFA. Estimation of the PPP split ratio of P. chrysogenum at a growth rate of 0.02 h(-1) yielded comparable values for the gluconate tracer method and the 13C-based MFA method, 51.8% and 51.1%, respectively. A sensitivity analysis of the estimated PPP split ratios showed that the 95% confidence interval was almost threefold smaller for the gluconate tracer method than for the 13C-based MFA method (40.0 to 63.5% and 46.0 to 56.5%, respectively). From these results we concluded that the gluconate tracer method permits accurate determination of the PPP split ratio but provides no information about the remaining cellular metabolism, while the 13C-based MFA method permits estimation of multiple fluxes but provides a less accurate estimate of the PPP split ratio.     

Bidirectional reaction steps in metabolic networks: II. Flux estimation and statistical analysis

Metabolic carbon labelling experiments enable a large amount of extracellular fluxes and intracellular carbon isotope enrichments to be measured. Since the relation between the measured quantities and the unknown intracellular metabolic fluxes is given by bilinear balance equations, flux determination from this data set requires the numerical solution of a nonlinear inverse problem. To this end, a general algorithm for flux estimation from metabolic carbon labelling experiments based on the least squares approach is developed in this contribution and complemented by appropriate tools for statistical analysis. The linearization technique usually applied for the computation of nonlinear confidence regions is shown to be inappropriate in the case of large exchange fluxes. For this reason a sophisticated compactification transformation technique for nonlinear statistical analysis is developed. Statistical analysis is then performed by computing appropriate statistical quality measures like output sensitivities, parameter sensitivities and the parameter covariance matrix. This allows one to determine the order of magnitude of exchange fluxes in most practical situations. An application study with a large data set from lysine-producing Corynebacterium glutamicum demonstrates the power and limitations of the carbon-labelling technique. It is shown that all intracellular fluxes in central metabolism can be quantitated without assumptions on intracellular energy yields. At the same time several exchange fluxes are determined which is invaluable information for metabolic engineering. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 118-135, 1997.     

A transient isotopic labeling methodology for 13C metabolic flux analysis of photoautotrophic microorganisms

Metabolic flux analysis is increasingly recognized as an integral component of systems biology. However, techniques for experimental measurement of system-wide metabolic fluxes in purely photoautotrophic systems (growing on CO(2) as the sole carbon source) have not yet been developed due to the unique problems posed by such systems. In this paper, we demonstrate that an approach that balances positional isotopic distributions transiently is the only route to obtaining system-wide metabolic flux maps for purely autotrophic metabolism. The outlined transient (13)C-MFA methodology enables measurement of fluxes at a metabolic steady-state, while following changes in (13)C-labeling patterns of metabolic intermediates as a function of time, in response to a step-change in (13)C-label input. We use mathematical modeling of the transient isotopic labeling patterns of central intermediates to assess various experimental requirements for photoautotrophic MFA. This includes the need for intracellular metabolite concentration measurements and isotopic labeling measurements as a function of time. We also discuss photobioreactor design and operation in order to measure fluxes under precise environmental conditions. The transient MFA technique can be used to measure and compare fluxes under different conditions of light intensity, nitrogen sources or compare strains with various mutations or gene deletions and additions.     

A constraint-based model analysis of the metabolic consequences of increased NADPH oxidation in Saccharomyces cerevisiae

Controlling the amounts of redox cofactors to manipulate metabolic fluxes is emerging as a useful approach to optimizing byproduct yields in yeast biotechnological processes. Redox cofactors are extensively interconnected metabolites, so predicting metabolite patterns is challenging and requires in-depth knowledge of how the metabolic network responds to a redox perturbation. Our aim was to analyze comprehensively the metabolic consequences of increased cytosolic NADPH oxidation during yeast fermentation. Using a genetic device based on the overexpression of a modified 2,3-butanediol dehydrogenase catalyzing the NADPH-dependent reduction of acetoin into 2,3-butanediol, we increased the NADPH demand to between 8 and 40-fold the anabolic demand. We developed (i) a dedicated constraint-based model of yeast fermentation and (ii) a constraint-based modeling method based on the dynamical analysis of mass distribution to quantify the in vivo contribution of pathways producing NADPH to the maintenance of redox homeostasis. We report that yeast responds to NADPH oxidation through a gradual increase in the flux through the PP and acetate pathways, providing 80% and 20% of the NADPH demand, respectively. However, for the highest NADPH demand, the model reveals a saturation of the PP pathway and predicts an exchange between NADH and NADPH in the cytosol that may be mediated by the glycerol-DHA futile cycle. We also reveal the contribution of mitochondrial shuttles, resulting in a net production of NADH in the cytosol, to fine-tune the NADH/NAD(+) balance. This systems level study helps elucidate the physiological adaptation of yeast to NADPH perturbation. Our findings emphasize the robustness of yeast to alterations in NADPH metabolism and highlight the role of the glycerol-DHA cycle as a redox valve, providing additional NADPH from NADH under conditions of very high demand.     

Genealogy profiling through strain improvement by using metabolic network analysis: metabolic flux genealogy of several generations of lysine-producing corynebacteria

A comprehensive approach of metabolite balancing, (13)C tracer studies, gas chromatography-mass spectrometry, matrix-assisted laser desorption ionization-time of flight mass spectrometry, and isotopomer modeling was applied for comparative metabolic network analysis of a genealogy of five successive generations of lysine-producing Corynebacterium glutamicum. The five strains examined (C. glutamicum ATCC 13032, 13287, 21253, 21526, and 21543) were previously obtained by random mutagenesis and selection. Throughout the genealogy, the lysine yield in batch cultures increased markedly from 1.2 to 24.9% relative to the glucose uptake flux. Strain optimization was accompanied by significant changes in intracellular flux distributions. The relative pentose phosphate pathway (PPP) flux successively increased, clearly corresponding to the product yield. Moreover, the anaplerotic net flux increased almost twofold as a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes to cover the increased demand for lysine formation; thus, the overall increase was a consequence of concerted regulation of C(3) carboxylation and C(4) decarboxylation fluxes. The relative flux through isocitrate dehydrogenase dropped from 82.7% in the wild type to 59.9% in the lysine-producing mutants. In contrast to the NADPH demand, which increased from 109 to 172% due to the increasing lysine yield, the overall NADPH supply remained constant between 185 and 196%, resulting in a decrease in the apparent NADPH excess through strain optimization. Extrapolated to industrial lysine producers, the NADPH supply might become a limiting factor. The relative contributions of PPP and the tricarboxylic acid cycle to NADPH generation changed markedly, indicating that C. glutamicum is able to maintain a constant supply of NADPH under completely different flux conditions. Statistical analysis by a Monte Carlo approach revealed high precision for the estimated fluxes, underlining the fact that the observed differences were clearly strain specific.     

Changes of in vivo fluxes through central metabolic pathways during the production of nystatin by Streptomyces noursei in batch culture

The central carbon metabolism of the nystatin-producing strain Streptomyces noursei ATCC 11455 was evaluated by 13C-labelling experiments. A batch fermentation was examined during the idiophase by GC-MS measurements of the labelling patterns of amino acids in the biomass. The labelling patterns of the amino acids and calculated fluxes of the central metabolism showed that changes in the primary and secondary metabolisms occurred simultaneously. Changes in the profiles for the integrated fluxes showed a decreased flux through the pentose phosphate pathway and an increased flux in the tricarboxylic acid cycle relative to the glucose uptake rate when the culture entered a phase with reduced specific growth rate and enhanced nystatin yield. The flux through the pentose phosphate pathway seemed to be adjusted according to the NADPH requirement during the different phases of the batch fermentation.     

Genome-scale metabolic flux analysis of Streptomyces lividans growing on a complex medium

Constraint-based metabolic modeling comprises various excellent tools to assess experimentally observed phenotypic behavior of micro-organisms in terms of intracellular metabolic fluxes. In combination with genome-scale metabolic networks, micro-organisms can be investigated in much more detail and under more complex environmental conditions. Although complex media are ubiquitously applied in industrial fermentations and are often a prerequisite for high protein secretion yields, such multi-component conditions are seldom investigated using genome-scale flux analysis. In this paper, a systematic and integrative approach is presented to determine metabolic fluxes in Streptomyces lividans TK24 grown on a nutritious and complex medium. Genome-scale flux balance analysis and randomized sampling of the solution space are combined to extract maximum information from exometabolome profiles. It is shown that biomass maximization cannot predict the observed metabolite production pattern as such. Although this cellular objective commonly applies to batch fermentation data, both input and output constraints are required to reproduce the measured biomass production rate. Rich media hence not necessarily lead to maximum biomass growth. To eventually identify a unique intracellular flux vector, a hierarchical optimization of cellular objectives is adopted. Out of various tested secondary objectives, maximization of the ATP yield per flux unit returns the closest agreement with the maximum frequency in flux histograms. This unique flux estimation is hence considered as a reasonable approximation for the biological fluxes. Flux maps for different growth phases show no active oxidative part of the pentose phosphate pathway, but NADPH generation in the TCA cycle and NADPH transdehydrogenase activity are most important in fulfilling the NADPH balance. Amino acids contribute to biomass growth by augmenting the pool of available amino acids and by boosting the TCA cycle, particularly when using glutamate and aspartate. Depletion of glutamate and aspartate causes a distinct shift in fluxes of the central carbon and nitrogen metabolism. In the current work, hurdles encountered in flux analysis at a genome-scale level are addressed using hierarchical flux balance analysis and uniform sampling of the constrained solution space. This general framework can now be adopted in further studies of S. lividans, e.g., as a host for heterologous protein production.     

A guide to metabolic flux analysis in metabolic engineering: Methods,tools and applications

The field of metabolic engineering is primarily concerned with improving the biological production of value-added chemicals, fuels and pharmaceuticals through the design, construction and optimization of metabolic pathways, redirection of intracellular fluxes, and refinement of cellular properties relevant for industrial bioprocess implementation. Metabolic network models and metabolic fluxes are central concepts in metabolic engineering, as was emphasized in the first paper published in this journal, “Metabolic fluxes and metabolic engineering” (Metabolic Engineering, 1: 1–11, 1999). In the past two decades, a wide range of computational, analytical and experimental approaches have been developed to interrogate the capabilities of biological systems through analysis of metabolic network models using techniques such as flux balance analysis (FBA), and quantify metabolic fluxes using constrained-based modeling approaches such as metabolic flux analysis (MFA) and more advanced experimental techniques based on the use of stable-isotope tracers, i.e. ¹³C-metabolic flux analysis (¹³C-MFA). In this review, we describe the basic principles of metabolic flux analysis, discuss current best practices in flux quantification, highlight potential pitfalls and alternative approaches in the application of these tools, and give a broad overview of pragmatic applications of flux analysis in metabolic engineering practice.     

Determining Actinobacillus succinogenes metabolic pathways and fluxes by NMR and GC-MS analyses of 13C-labeled metabolic product isotopomers

Actinobacillus succinogenes is a promising candidate for industrial succinate production. However, in addition to producing succinate, it also produces formate and acetate. To understand carbon flux distribution to succinate and alternative products we fed A. succinogenes [1-(13)C]glucose and analyzed the resulting isotopomers of excreted organic acids, proteinaceous amino acids, and glycogen monomers by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. The isotopomer data, together with the glucose consumption and product formation rates and the A. succinogenes biomass composition, were supplied to a metabolic flux model. Oxidative pentose phosphate pathway flux supplied, at most, 20% of the estimated NADPH requirement for cell growth. The model indicated that NADPH was instead produced primarily by the conversion of NADH to NADPH by transhydrogenase and/or by NADP-dependent malic enzyme. Transhydrogenase activity was detected in A. succinogenes cell extracts, as were formate and pyruvate dehydrogenases, which the model suggested were contributing to NADH production. Malic enzyme activity was also detected in cell extracts, consistent with the flux analysis results. Labeling patterns in amino acids and organic acids showed that oxaloacetate and malate were being decarboxylated to pyruvate. These are the first in vivo experiments to show that the partitioning of flux between succinate and alternative fermentation products can occur at multiple nodes in A. succinogenes. The implications for designing effective metabolic engineering strategies to increase A. succinogenes succinate production are discussed.