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Flux Analysis (flux + analysis)
Kinds of Flux Analysis Selected AbstractsDetermination of Biomass Composition of Catharanthusroseus Hairy Roots for Metabolic Flux AnalysisBIOTECHNOLOGY PROGRESS, Issue 6 2006Ganesh Sriram 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. [source] Flux analysis gets faster, less expensive, and more relevantBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2008Article first published online: 19 DEC 200 No abstract is available for this article. [source] Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockoutBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Chen Yang Abstract The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose. The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E. coli. The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U- 13C6]glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol. Significant activity of PEP carboxykinase was identified in wild-type E. coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux. Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt. The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined. Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E. coli are discussed. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 129,144, 2003. [source] Non-invasive quantification of endogenous root auxin transport using an integrated flux microsensor techniqueTHE PLANT JOURNAL, Issue 6 2010Eric S. McLamore Summary Indole-3-acetic acid (IAA) is a primary phytohormone that regulates multiple aspects of plant development. Because polar transport of IAA is an essential determinant of organogenesis and dynamic tropic growth, methods to monitor IAA movement in vivo are in demand. A self-referencing electrochemical microsensor was optimized to non-invasively measure endogenous IAA flux near the surface of Zea mays roots without the addition of exogenous IAA. Enhanced sensor surface modification, decoupling of acquired signals, and integrated flux analyses were combined to provide direct, real time quantification of endogenous IAA movement in B73 maize inbred and brachytic2 (br2) auxin transport mutant roots. BR2 is localized in epidermal and hypodermal tissues at the root apex. br2 roots exhibit reduced shootward IAA transport at the root apex in radiotracer experiments and reduced gravitropic growth. IAA flux data indicates that maximal transport occurs in the distal elongation zone of maize roots, and net transport in/out of br2 roots was decreased compared to B73. Integration of short term real time flux data in this zone revealed oscillatory patterns, with B73 exhibiting shorter oscillatory periods and greater amplitude than br2. IAA efflux and influx were inhibited using 1- N -naphthylphthalamic acid (NPA), and 2-naphthoxyacetic acid (NOA), respectively. A simple harmonic oscillation model of these data produced a correlation between modeled and measured values of 0.70 for B73 and 0.69 for br2. These results indicate that this technique is useful for real-time IAA transport monitoring in surface tissues and that this approach can be performed simultaneously with current live imaging techniques. [source] 13C-Labeled metabolic flux analysis of a fed-batch culture of elutriated Saccharomyces cerevisiaeFEMS YEAST RESEARCH, Issue 4 2007Roeland Costenoble Abstract 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. [source] Mechanistic understanding of the fermentative L -glutamic acid overproduction by Corynebacterium glutamicum through combined metabolic flux profiling and transmembrane transport characteristicsJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 12 2004Theodora Tryfona Abstract Since the 1950s when Micrococcus glutamicus later renamed Corynebacterium glutamicum was discovered, the production of amino acids by fermentative methods has become an important aspect of industrial microbiology. Numerous studies to understand and improve the metabolic conditions leading to amino acid overproduction have been carried out. Most amino acids are currently produced by use of mutants that contain combinations of auxotrophic and regulatory mutations. L -Glutamic acid is the amino acid produced in the greatest quantities (106 tonnes per year) and Corynebacteria are central to its industrial production. However, further improvements to strain performance are difficult to obtain by empirical optimization and a more rational approach is required. The use of metabolic flux analysis provides valuable information regarding bottlenecks in the formation of desired metabolites. Such techniques have found application in elucidating flux control, provided insight into metabolic network function and developed methods to amplify or redirect fluxes in engineered bioprocesses. Hence, branch points in biosynthesis, precursor supply in fuelling reactions and export of metabolites can be manipulated, resulting in high glutamic acid overproduction by Corynebacterium glutamicum fermentations. In this review, in addition to reviewing the state of play in metabolic flux analysis for glutamate overproduction, the metabolic pathways involved in the production of L -glutamic acid, the mechanisms mediating its efflux and secretion as well as their manipulation to achieve higher glutamate production, are discussed. The link between metabolic flux and transmembrane transport of glutamic acid are also considered. Copyright © 2004 Society of Chemical Industry [source] Revealing metabolic phenotypes in plants: inputs from NMR analysisBIOLOGICAL REVIEWS, Issue 1 2005R. G. Ratcliffe ABSTRACT Assessing the performance of the plant metabolic network, with its varied biosynthetic capacity and its characteristic subcellular compartmentation, remains a considerable challenge. The complexity of the network is such that it is not yet possible to build large-scale predictive models of the fluxes it supports, whether on the basis of genomic and gene expression analysis or on the basis of more traditional measurements of metabolites and their interconversions. This limits the agronomic and biotechnological exploitation of plant metabolism, and it undermines the important objective of establishing a rational metabolic engineering strategy. Metabolic analysis is central to removing this obstacle and currently there is particular interest in harnessing high-throughput and/or large-scale analyses to the task of defining metabolic phenotypes. Nuclear magnetic resonance (NMR) spectroscopy contributes to this objective by providing a versatile suite of analytical techniques for the detection of metabolites and the fluxes between them. The principles that underpin the analysis of plant metabolism by NMR are described, including a discussion of the measurement options for the detection of metabolites in vivo and in vitro, and a description of the stable isotope labelling experiments that provide the basis for metabolic flux analysis. Despite a relatively low sensitivity, NMR is suitable for high-throughput system-wide analyses of the metabolome, providing methods for both metabolite fingerprinting and metabolite profiling, and in these areas NMR can contribute to the definition of plant metabolic phenotypes that are based on metabolic composition. NMR can also be used to investigate the operation of plant metabolic networks. Labelling experiments provide information on the operation of specific pathways within the network, and the quantitative analysis of steady-state labelling experiments leads to the definition of large-scale flux maps for heterotrophic carbon metabolism. These maps define multiple unidirectional fluxes between branch-points in the metabolic network, highlighting the existence of substrate cycles and discriminating in favourable cases between fluxes in the cytosol and plastid. Flux maps can be used to define a functionally relevant metabolic phenotype and the extensive application of such maps in microbial systems suggests that they could have important applications in characterising the genotypes produced by plant genetic engineering. [source] Stoichiometric model and metabolic flux analysis for Leptospirillum ferrooxidansBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010M.P. Merino Abstract A metabolic model for Leptospirillum ferrooxidans was developed based on the genomic information of an analogous iron oxidizing bacteria and on the pathways of ferrous iron oxidation, nitrogen and CO2 assimilation based on experimental evidence for L. ferrooxidans found in the literature. From this metabolic reconstruction, a stoichiometric model was built, which includes 86 reactions describing the main catabolic and anabolic aspects of its metabolism. The model obtained has 2 degrees of freedom, so two external fluxes were estimated to achieve a determined and observable system. By using the external oxygen consumption rate and the generation flux biomass as input data, a metabolic flux map with a distribution of internal fluxes was obtained. The results obtained were verified with experimental data from the literature, achieving a very good prediction of the metabolic behavior of this bacterium at steady state. Biotechnol. Bioeng. 2010;107:696,706. © 2010 Wiley Periodicals, Inc. [source] Quantitative physiology of Pichia pastoris during glucose-limited high-cell density fed-batch cultivation for recombinant protein productionBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Jan Heyland Abstract Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high-cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed-batch cultivations, very high-cell densities were reached (more than 200,gCDW,L,1) resulting in a recombinant protein titer of about 6.5,g,L,1. To investigate the impact of recombinant protein production and high-cell density fermentation on the metabolism of P. pastoris, we used 13C-tracer-based metabolic flux analysis in batch and fed-batch experiments. At a controlled growth rate of 0.12,h,1 in fed-batch experiments an increased TCA cycle flux of 1.1,mmol,g,1,h,1 compared to 0.7,mmol,g,1,h,1 for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development. Biotechnol. Bioeng. 2010;107: 357,368. © 2010 Wiley Periodicals, Inc. [source] Metabolic flux analysis for recombinant protein production by Pichia pastoris using dual carbon sources: Effects of methanol feeding rateBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2010Eda Ēelik Abstract The intracellular metabolic fluxes through the central carbon pathways in the bioprocess for recombinant human erythropoietin (rHuEPO) production by Pichia pastoris (Mut+) were calculated to investigate the metabolic effects of dual carbon sources (methanol/sorbitol) and the methanol feed rate, and to obtain a deeper understanding of the regulatory circuitry of P. pastoris, using the established stoichiometry-based model containing 102 metabolites and 141 reaction fluxes. Four fed-batch operations with (MS-) and without (M-) sorbitol were performed at three different constant specific growth rates (h,1), and denoted as M-0.03, MS-0.02, MS-0.03, and MS-0.04. Considering the methanol consumption pathway, the M-0.03 and MS-0.02 conditions produced similar effects and had >85% of formaldehyde flux towards the assimilatory pathway. In contrast, the use of the dual carbon source condition generated a shift in metabolism towards the dissimilatory pathway that corresponded to the shift in dilution rate from MS-0.03 to MS-0.04, indicating that the methanol feed exceeded the metabolic requirements at the higher µ0. Comparing M-0.03 and MS-0.03 conditions, which had the same methanol feeding rates, sorbitol addition increased the rHuEPO synthetic flux 4.4-fold. The glycolysis, gluconeogenesis, and PPP pathways worked uninterruptedly only at MS-0.02 condition. PPP and TCA cycles worked with the highest disturbances at MS-0.04 condition, which shows the stress of increased feeding rates of methanol on cell metabolism. Biotechnol. Bioeng. 2010; 105: 317,329. © 2009 Wiley Periodicals, Inc. [source] Aldehyde,alcohol dehydrogenase and/or thiolase overexpression coupled with CoA transferase downregulation lead to higher alcohol titers and selectivity in Clostridium acetobutylicum fermentationsBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009Ryan Sillers Abstract Metabolic engineering (ME) of Clostridium acetobutylicum has led to increased solvent (butanol, acetone, and ethanol) production and solvent tolerance, thus demonstrating that further efforts have the potential to create strains of industrial importance. With recently developed ME tools, it is now possible to combine genetic modifications and thus implement more advanced ME strategies. We have previously shown that antisense RNA (asRNA)-based downregulation of CoA transferase (CoAT, the first enzyme in the acetone-formation pathway) results in increased butanol to acetone selectivity, but overall reduced butanol yields and titers. In this study the alcohol/aldehyde dehydrogenase (aad) gene (encoding the bifunctional protein AAD responsible for butanol and ethanol production from butyryl-CoA and acetyl-CoA, respectively) was expressed from the phosphotransbutyrylase (ptb) promoter to enhance butanol formation and selectivity, while CoAT downregulation was used to minimize acetone production. This led to early production of high alcohol (butanol plus ethanol) titers, overall solvent titers of 30 g/L, and a higher alcohol/acetone ratio. Metabolic flux analysis revealed the likely depletion of butyryl-CoA. In order to increase then the flux towards butyryl-CoA, we examined the impact of thiolase (THL, thl) overexpression. THL converts acetyl-CoA to acetoacetyl-CoA, the first step of the pathway from acetyl-CoA to butyryl-CoA, and thus, combining thl overexpression with aad overexpression decreased, as expected, acetate and ethanol production while increasing acetone and butyrate formation. thl overexpression in strains with asRNA CoAT downregulation did not significantly alter product formation thus suggesting that a more complex metabolic engineering strategy is necessary to enhance the intracellular butyryl-CoA pool and reduce the acetyl-CoA pool in order to achieve improved butanol titers and selectivity. Biotechnol. Bioeng. 2009;102: 38,49. © 2008 Wiley Periodicals, Inc. [source] An elementary metabolite unit (EMU) based method of isotopically nonstationary flux analysis,BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2008Jamey D. Young Abstract Nonstationary metabolic flux analysis (NMFA) is at present a very computationally intensive exercise, especially for large reaction networks. We applied elementary metabolite unit (EMU) theory to NMFA, dramatically reducing computational difficulty. We also introduced block decoupling, a new method that systematically and comprehensively divides EMU systems of equations into smaller subproblems to further reduce computational difficulty. These improvements led to a 5000-fold reduction in simulation times, enabling an entirely new and more complicated set of problems to be analyzed with NMFA. We simulated a series of nonstationary and stationary GC/MS measurements for a large E. coli network that was then used to estimate parameters and their associated confidence intervals. We found that fluxes could be successfully estimated using only nonstationary labeling data and external flux measurements. Addition of near-stationary and stationary time points increased the precision of most parameters. Contrary to prior reports, the precision of nonstationary estimates proved to be comparable to the precision of estimates based solely on stationary data. Finally, we applied EMU-based NMFA to experimental nonstationary measurements taken from brown adipocytes and successfully estimated fluxes and some metabolite concentrations. By using NFMA instead of traditional MFA, the experiment required only 6 h instead of 50 (the time necessary for most metabolite labeling to reach 99% of isotopic steady state). Biotechnol. Bioeng. 2008;99: 686,699. © 2007 Wiley Periodicals, Inc. [source] Analysis of Escherichia coli anaplerotic metabolism and its regulation mechanisms from the metabolic responses to altered dilution rates and phosphoenolpyruvate carboxykinase knockoutBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2003Chen Yang Abstract The gluconeogenic phosphoenolpyruvate (PEP) carboxykinase is active in Escherichia coli during its growth on glucose. The present study investigated the influence of growth rates and PEP carboxykinase knockout on the anaplerotic fluxes in E. coli. The intracellular fluxes were determined using the complementary methods of flux ratio analysis and metabolic flux analysis based on [U- 13C6]glucose labeling experiments and 2D nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids and glycerol. Significant activity of PEP carboxykinase was identified in wild-type E. coli, and the ATP dissipation for the futile cycling via this reaction accounted for up to 8.2% of the total energy flux. Flux analysis of pck deletion mutant revealed that abolishment of PEP carboxykinase activity resulted in a remarkably reduced flux through the anaplerotic PEP carboxylase and the activation of the glyoxylate shunt, with 23% of isocitrate found being channeled in the glyoxylate shunt. The changes in intracellular metabolite concentrations and specific enzyme activities associated with different growth rates and pck deletion, were also determined. Combining the measurement data of in vivo fluxes, metabolite concentrations and enzyme activities, the in vivo regulations of PEP carboxykinase flux, PEP carboxylation, and glyoxylate shunt in E. coli are discussed. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 84: 129,144, 2003. [source] Cumulative bondomers: A new concept in flux analysis from 2D [13C,1H] COSY NMR dataBIOTECHNOLOGY & BIOENGINEERING, Issue 7 2002Wouter A. van Winden Abstract A well-established way of determining metabolic fluxes is to measure 2D [13C,1H] COSY NMR spectra of components of biomass grown on uniformly 13C-labeled carbon sources. When using the entire set of measured data to simultaneously determine all fluxes in a proposed metabolic network model, the 13C-labeling distribution in all measured compounds has to be simulated. This requires very large sets of isotopomer or cumomer balances. This article introduces the new concept of bondomers; entities that only vary in the numbers and positions of C,C bonds that have remained intact since the medium substrate molecule entered the metabolism. Bondomers are shown to have many analogies to isotopomers. One of these is that bondomers can be transformed to cumulative bondomers, just like isotopomers can be transformed to cumomers. Similarly to cumomers, cumulative bondomers allow an analytical solution of the entire set of balances describing a metabolic network. The main difference is that cumulative bondomer models are considerably smaller than corresponding cumomer models. This saves computational time, allows easier identifiability analysis, and yields new insights in the information content of 2D [13C,1H] COSY NMR data. We illustrate the theoretical concepts by means of a realistic example of the glycolytic and pentose phosphate pathways. The combinations of 2D [13C,1H] COSY NMR data that allow identification of all metabolic fluxes in these pathways are analyzed, and it is found that the NMR data contain less information than was previously expected. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 731,745, 2002. [source] Correcting mass isotopomer distributions for naturally occurring isotopesBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2002Wouter A. Van Winden In one method of metabolic flux analysis, simulated mass spectrometry data is fitted to measured mass distributions of metabolites that are isolated from cultures with defined feeding of 13C-labeled substrates. Doing so, simulated mass distributions must be corrected for the presence of naturally occurring isotopes. A method that was recently introduced for this purpose consists of consecutive correction steps for each isotope of each element in the considered compound. Here we show that all isotopes of each individual element must, however, be corrected in one single step. Furthermore, it is shown that the source of information with respect to isotopic compositions of the elements needs to be chosen with care. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 477,479, 2002. [source] Modeling with a view to target identification in metabolic engineering: A critical evaluation of the available toolsBIOTECHNOLOGY PROGRESS, Issue 2 2010Jo Maertens Abstract The state of the art tools for modeling metabolism, typically used in the domain of metabolic engineering, were reviewed. The tools considered are stoichiometric network analysis (elementary modes and extreme pathways), stoichiometric modeling (metabolic flux analysis, flux balance analysis, and carbon modeling), mechanistic and approximative modeling, cybernetic modeling, and multivariate statistics. In the context of metabolic engineering, one should be aware that the usefulness of these tools to optimize microbial metabolism for overproducing a target compound depends predominantly on the characteristic properties of that compound. Because of their shortcomings not all tools are suitable for every kind of optimization; issues like the dependence of the target compound's synthesis on severe (redox) constraints, the characteristics of its formation pathway, and the achievable/desired flux towards the target compound should play a role when choosing the optimization strategy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Study of stationary phase metabolism via isotopomer analysis of amino acids from an isolated proteinBIOTECHNOLOGY PROGRESS, Issue 1 2010Afshan S. Shaikh Abstract 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 [source] Invariability of central metabolic flux distribution in Shewanella oneidensis MR-1 under environmental or genetic perturbationsBIOTECHNOLOGY PROGRESS, Issue 5 2009Yinjie J. Tang Abstract An environmentally important bacterium with versatile respiration, Shewanella oneidensis MR-1, displayed significantly different growth rates under three culture conditions: minimal medium (doubling time ,3 h), salt stressed minimal medium (doubling time ,6 h), and minimal medium with amino acid supplementation (doubling time ,1.5 h). 13C-based metabolic flux analysis indicated that fluxes of central metabolic reactions remained relatively constant under the three growth conditions, which is in stark contrast to the reported significant changes in the transcript and metabolite profiles under various growth conditions. Furthermore, 10 transposon mutants of S. oneidensis MR-1 were randomly chosen from a transposon library and their flux distributions through central metabolic pathways were revealed to be identical, even though such mutational processes altered the secondary metabolism, for example, glycine and C1 (5,10-Me-THF) metabolism. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] On-line adaptive metabolic flux analysis: Application to PHB production by mixed microbial culturesBIOTECHNOLOGY PROGRESS, Issue 2 2009Joćo Dias Abstract In this work, an algorithm for on-line adaptive metabolic flux analysis (MFA) is proposed and applied to polyhydroxybutyrate (PHB) production by mixed microbial cultures (MMC). In this process, population dynamics constitutes an important source of perturbation to MFA calculations because some stoichiometric and energetic parameters of the underlying metabolic network are continuously changing over time. The proposed algorithm is based on the application of the observer-based estimator (OBE) to the central MFA equation, whereby the role of the OBE is to force the accumulation of intracellular metabolites to converge to zero by adjusting the values of unknown network parameters. The algorithm was implemented in a reactor equipped with on-line analyses of dissolved oxygen and carbon dioxide through respirometric and titrimetric measurements. The oxygen and carbon dioxide fluxes were measured directly, whereas acetate, PHB, and sludge production fluxes were estimated indirectly using a projection of latent structures model calibrated a priori with off-line measurements. The algorithm was implemented in a way that the network parameters associated with biosynthesis were adjusted on-line. The algorithm proofed to converge exponentially with the steady state error always below 1 mmol/L. The estimated fluxes passed the consistency index test for experimental error variances as low as 1%. The comparison of measured and estimated respiratory coefficient and of the theoretical and estimated yield of sludge on acetate further confirmed the metabolic consistency of the parameters that were estimated on-line. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Determination of Biomass Composition of Catharanthusroseus Hairy Roots for Metabolic Flux AnalysisBIOTECHNOLOGY PROGRESS, Issue 6 2006Ganesh Sriram 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. [source] Application of Multivariate Analysis to Optimize Function of Cultured HepatocytesBIOTECHNOLOGY PROGRESS, Issue 2 2003Christina Chan Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells, including the development of bioartificial liver (BAL) devices. To characterize intermediary metabolism in the hepatocytes, metabolic flux analysis (MFA) was applied to elucidate the changes in intracellular pathway fluxes of primary rat hepatocytes exposed to human plasma and to provide a comprehensive snapshot of the hepatic metabolic profile. In the current study, the combination of preconditioning and plasma supplementation produced distinct metabolic states. Combining the metabolic flux distribution obtained by MFA with methodologies such as Fisher discriminant analysis (FDA) and partial least squares or projection to latent structures (PLS) provided insights into the underlying structure and causal relationship within the data. With the aid of these analyses, patterns in the cellular response of the hepatocytes that contributed to the separation of the different hepatic states were identified. Of particular interest was the recognition of distal pathways that strongly correlated with a particular hepatic function. The hepatic functions investigated were intracellular triglyceride accumulation and urea production. This study illustrates a framework for optimizing hepatic function and a possibility of identifying potential targets for improving hepatic functions. [source] Improving Glucose and Glutamine Metabolism of Human HEK 293 and Trichoplusiani Insect Cells Engineered To Express a Cytosolic Pyruvate Carboxylase EnzymeBIOTECHNOLOGY PROGRESS, Issue 1 2003Cynthia B. Elias Metabolic engineering has been defined as a directed improvement of product formation or cellular properties by modification of specific biochemical pathways or introduction of new enzymatic reactions by recombinant DNA technology. The use of metabolic flux analysis (MFA) has helped in the understanding of the key limitation in the metabolic pathways of cultured animal cells. The MFA of the major nutrients glucose and glutamine showed that the flux of glucose to the TCA cycle and its subsequent utilization is limited as a result of the lack of certain key enzymes in the pathway. One of the key enzymes controlling this flux is pyruvate carboxylase. Introduction of this enzyme into mammalian cells has been shown to improve the utilization of glucose and limit the production of lactate and ammonia, which are deleterious to cell growth. In the present work a yeast pyruvate carboxylase gene has been introduced into mammalian (HEK 293) and insect ( Trichoplusiani High-Five) cells, resulting in the cytosolic expression of the enzyme. In both cases the resulting transfected cells were able to utilize glucose and glutamine more efficiently and produce lower amounts of lactate and ammonia. Differences in the amino acid utilization pattern were also observed, indicating changes in the basic metabolism of the cells. The performance of the transfected cells as expression systems for adenovirus and baculovirus vectors, respectively, has also been examined. The results obtained and their impact on the process development for protein and viral vector production are discussed. [source] | ||||||||||