Intracellular Reaction Rates Research Articles

Successful downsizing for high-throughput ¹³C-MFA applications.

(13)C label-based metabolic flux analysis is a powerful technique for the determination of intracellular reaction rates and is used in such different research fields as quantitative physiology, metabolic engineering, and systems biology. Metabolic fluxes can be determined at high quality using (pseudo)-steady-state cultures and advanced mathematical models for data interpretation. Here, we describe a protocol for parallel metabolic flux analysis that consists of downsized microbial (yeast) cultivation, miniaturized sample preparation, and semiautomated analytics and data evaluation. With this protocol dozens of metabolic flux analyses can be carried out in 1 week, thereby enabling for example the analysis of genetic and environmental perturbations on the operation of metabolic networks.

Metabolic reaction network of Pichia pastoris with glycosylation reactions: Flux analysis for erythropoietin production

AbstractBACKGROUNDBiochemical reaction network of Pichia pastoris was improved by including N‐ glycosylation pathway reactions to determine the intracellular reaction rates for glycosylated protein production.RESULTSWhen co‐substrate sorbitol was replaced with mannitol, 12‐fold increase in erythropoietin flux was obtained as a result of 1.2‐ and 2.4‐fold increase in glucose‐6‐phosphate (G6P) formation from fructose‐6‐phosphate (F6P) and 3‐phospho‐D‐glycerate (3PG) formation from glyceraldehyde‐3‐phosphate (G3P), respectively. Thus, to analyse the fluxes around F6P and the fluxes of the glycosylation pathway reactions in depth, the biochemical reaction network of P. pastoris containing 204 reactions and 129 metabolites was developed and used. The flux analysis for glycosylated erythropoietin production with three glycan residues having 12 mannoses (EPO‐Man12) reveals that the fluxes from G3P and guanosine 5′‐diphosphate (GDP) to F6P synthesis were 1.6‐ and 3‐fold higher than that of the fluxes determined for erythropoietin production with no glycans (EPO), respectively. Furthermore, the results of theoretical data‐based overproduction capacity show that the fluxes of EPO‐Man12 and partially humanized EPO with three glycan residues having five mannoses (EPO‐Man5) were 3.6 and 3.8 µmol gDCW‐1 h‐1, respectively, when the methanol and mannitol uptake rates were 2.10 mmol gDCW‐1 h‐1 and 1.49 mmol gDCW‐1 h‐1. For glycoprotein production, fluxes around F6P were found to be important as UDP‐N‐acetyl‐α‐D‐glucosamine (UDP‐GlcNAc) and guanosine 5′‐diphosphate‐D‐Mannose (GDP‐Man) were produced from F6P. Thus the co‐substrate mannitol does not repress AOX promoter but is easily converted to F6P, enhancing glycoprotein production.CONCLUSIONA metabolic reaction network for P. pastoris with glycosylation reactions is proposed for the determination of fluxes for glycoprotein production by P. pastoris. © 2013 Society of Chemical Industry

Metabolic flux analysis of Cyanothece sp. ATCC 51142 under mixotrophic conditions

Cyanobacteria are a group of photosynthetic prokaryotes capable of utilizing solar energy to fix atmospheric carbon dioxide to biomass. Despite several "proof of principle" studies, low product yield is an impediment in commercialization of cyanobacteria-derived biofuels. Estimation of intracellular reaction rates by (13)C metabolic flux analysis ((13)C-MFA) would be a step toward enhancing biofuel yield via metabolic engineering. We report (13)C-MFA for Cyanothece sp. ATCC 51142, a unicellular nitrogen-fixing cyanobacterium, known for enhanced hydrogen yield under mixotrophic conditions. Rates of reactions in the central carbon metabolism under nitrogen-fixing and -non-fixing conditions were estimated by monitoring the competitive incorporation of (12)C and (13)C from unlabeled CO2 and uniformly labeled glycerol, respectively, into terminal metabolites such as amino acids. The observed labeling patterns suggest mixotrophic growth under both the conditions, with a larger fraction of unlabeled carbon in nitrate-sufficient cultures asserting a greater contribution of carbon fixation by photosynthesis and an anaplerotic pathway. Indeed, flux analysis complements the higher growth observed under nitrate-sufficient conditions. On the other hand, the flux through the oxidative pentose phosphate pathway and tricarboxylic acid cycle was greater in nitrate-deficient conditions, possibly to supply the precursors and reducing equivalents needed for nitrogen fixation. In addition, an enhanced flux through fructose-6-phosphate phosphoketolase possibly suggests the organism's preferred mode under nitrogen-fixing conditions. The (13)C-MFA results complement the reported predictions by flux balance analysis and provide quantitative insight into the organism's distinct metabolic features under nitrogen-fixing and -non-fixing conditions.

Flux-p: automating metabolic flux analysis.

Quantitative knowledge of intracellular fluxes in metabolic networks is invaluable for inferring metabolic system behavior and the design principles of biological systems. However, intracellular reaction rates can not often be calculated directly but have to be estimated; for instance, via 13C-based metabolic flux analysis, a model-based interpretation of stable carbon isotope patterns in intermediates of metabolism. Existing software such as FiatFlux, OpenFLUX or 13CFLUX supports experts in this complex analysis, but requires several steps that have to be carried out manually, hence restricting the use of this software for data interpretation to a rather small number of experiments. In this paper, we present Flux-P as an approach to automate and standardize 13C-based metabolic flux analysis, using the Bio-jETI workflow framework. Exemplarily based on the FiatFlux software, it demonstrates how services can be created that carry out the different analysis steps autonomously and how these can subsequently be assembled into software workflows that perform automated, high-throughput intracellular flux analysis of high quality and reproducibility. Besides significant acceleration and standardization of the data analysis, the agile workflow-based realization supports flexible changes of the analysis workflows on the user level, making it easy to perform custom analyses.

Agent-based simulation of reactions in the crowded and structured intracellular environment: Influence of mobility and location of the reactants

BackgroundIn this paper we apply a novel agent-based simulation method in order to model intracellular reactions in detail. The simulations are performed within a virtual cytoskeleton enriched with further crowding elements, which allows the analysis of molecular crowding effects on intracellular diffusion and reaction rates. The cytoskeleton network leads to a reduction in the mobility of molecules. Molecules can also unspecifically bind to membranes or the cytoskeleton affecting (i) the fraction of unbound molecules in the cytosol and (ii) furthermore reducing the mobility. Binding of molecules to intracellular structures or scaffolds can in turn lead to a microcompartmentalization of the cell. Especially the formation of enzyme complexes promoting metabolic channeling, e.g. in glycolysis, depends on the co-localization of the proteins.ResultsWhile the co-localization of enzymes leads to faster reaction rates, the reduced mobility decreases the collision rate of reactants, hence reducing the reaction rate, as expected. This effect is most prominent in diffusion limited reactions. Furthermore, anomalous diffusion can occur due to molecular crowding in the cell. In the context of diffusion controlled reactions, anomalous diffusion leads to fractal reaction kinetics. The simulation framework is used to quantify and separate the effects originating from molecular crowding or the reduced mobility of the reactants. We were able to define three factors which describe the effective reaction rate, namely f diff for the diffusion effect, f volume for the crowding, and f access for the reduced accessibility of the molecules.ConclusionsMolecule distributions, reaction rate constants and structural parameters can be adjusted separately in the simulation allowing a comprehensive study of individual effects in the context of a realistic cell environment. As such, the present simulation can help to bridge the gap between in vivo and in vitro kinetics.

Integration of transcriptional inputs at promoters of the arabinose catabolic pathway.

BackgroundMost modelling efforts of transcriptional networks involve estimations of in vivo concentrations of components, binding affinities and reaction rates, derived from in vitro biochemical assays. These assays are difficult and in vitro measurements may not approximate actual in vivo conditions. Alternatively, changes in transcription factor activity can be estimated by using partially specified models which estimate the "hidden functions" of transcription factor concentration changes; however, non-unique solutions are a potential problem. We have applied a synthetic biology approach to develop reporters that are capable of measuring transcription factor activity in vivo in real time. These synthetic reporters are comprised of a constitutive promoter with an operator site for the specific transcription factor immediately downstream. Thus, increasing transcription factor activity is measured as repression of expression of the transcription factor reporter. Measuring repression instead of activation avoids the complications of non-linear interactions between the transcription factor and RNA polymerase which differs at each promoter.ResultsUsing these reporters, we show that a simple model is capable of determining the rules of integration for multiple transcriptional inputs at the four promoters of the arabinose catabolic pathway. Furthermore, we show that despite the complex and non-linear changes in cAMP-CRP activity in vivo during diauxic shift, the synthetic transcription factor reporters are capable of measuring real-time changes in transcription factor activity, and the simple model is capable of predicting the dynamic behaviour of the catabolic promoters.ConclusionsUsing a synthetic biology approach we show that the in vivo activity of transcription factors can be quantified without the need for measuring intracellular concentrations, binding affinities and reaction rates. Using measured transcription factor activity we show how different promoters can integrate common transcriptional inputs, resulting in distinct expression patterns. The data collected show that cAMP levels in vivo are dynamic and agree with observations showing that cAMP levels show a transient pulse during diauxic shift.

Comparative analysis of nitrite uptake and hemoglobin-nitrite reactions in erythrocytes: sorting out uptake mechanisms and oxygenation dependencies

Nitrite uptake into red blood cells (RBCs) precedes its intracellular reactions with hemoglobin (Hb) that forms nitric oxide (NO) during hypoxia. We investigated the uptake of nitrite and its reactions with Hb at different oxygen saturations (So(2)), using RBCs with (carp and rabbit) and without (hagfish and lamprey) anion exchanger-1 (AE1) in the membrane, with the aim to unravel the mechanisms and oxygenation dependencies of nitrite transport. Added nitrite rapidly diffused into the RBCs until equilibrium. The distribution ratio of nitrite across the membrane agreed with that expected from HNO(2) diffusion and AE1-mediated facilitated NO(2)(-) diffusion. Participation of HNO(2) diffusion was emphasized by rapid transmembrane nitrite equilibration also in the natural AE1 knockouts. Following the equilibration, nitrite was consumed by reacting with Hb, which created a continued inward diffusion controlled by intracellular reaction rates. Changes in nitrite uptake with So(2), pH, or species were accordingly explained by corresponding changes in reaction rates. In carp, nitrite uptake rates increased linearly with decreasing So(2) over the entire So(2) range. In rabbit, nitrite uptake rates were highest at intermediate So(2), producing a bell-shaped relationship with So(2). Nitrite consumption increased approximately 10-fold with a 1 unit decrease in pH, as expected from the involvement of protons in the reactions with Hb. The reaction of nitrite with deoxyhemoglobin was favored over that with oxyhemoglobin at intermediate So(2). We propose a model for RBC nitrite uptake that involves both HNO(2) diffusion and AE1-mediated transport and that explains both the present and previous (sometimes puzzling) results.

Metabolic networks in motion: 13 C‐based flux analysis

Many properties of complex networks cannot be understood from monitoring the components—not even when comprehensively monitoring all protein or metabolite concentrations—unless such information is connected and integrated through mathematical models. The reason is that static component concentrations, albeit extremely informative, do not contain functional information per se. The functional behavior of a network emerges only through the nonlinear gene, protein, and metabolite interactions across multiple metabolic and regulatory layers. I argue here that intracellular reaction rates are the functional end points of these interactions in metabolic networks, hence are highly relevant for systems biology. Methods for experimental determination of metabolic fluxes differ fundamentally from component concentration measurements; that is, intracellular reaction rates cannot be detected directly, but must be estimated through computer model-based interpretation of stable isotope patterns in products of metabolism.

A novel approach for on line monitoring of apoptotic cell shrinkage in individual live lymphocytes

The apoptotic process occurs asynchronically in most cell populations and its duration is variable. Therefore, the ability to continuously monitor the death process occurring in individual blood cells before, during and following apoptosis induction is crucial in the evaluation of the efficiency of pro- or anti-apoptotic drugs. We applied a kinetic approach by performing real time measurements of individual living cells. This approach is based on an easy and unique method for monitoring intracellular staining reaction, which accompanied early apoptotic cell shrinkage. The intracellular enzymatic reaction rates were determined by taking repeated, sequential measurements of fluorescence intensity of the same individual cells. These rates were found to correlate with the respective radii of the cells under different conditions, and to decrease following apoptosis induction. The ability to remeasure the same cell before and after apoptosis induction enabled the detection of specific individual lymphocytes, which were more susceptible or resistant to pro-apoptotic stimulus.

Bioreactor operation parameters as tools for metabolic regulations in fermentation processes: influence of pH conditions

The influence of controlled- and uncontrolled-pH conditions together with the initial pH on the product and by-product distributions and oxygen transfer characteristics, whereupon the process rate limitations in relation to the intracellular reaction rates were investigated in serine alkaline protease (SAP) fermentation process by recombinant Bacillus licheniformis carrying pHV1431 ::subC on a defined medium with the sole carbon source glucose in the pH range of 6.80–7.25 in batch bioreactors. Although the same amount of cell was produced at all the conditions in each type of operation, with the increase of initial pH the cell formation rates increased; moreover, uncontrolled-pH operation was favourable for the cell formation. The SAP synthesis rates and concentrations were higher at uncontrolled-pH operations. Among the fermentations, pH 0=7.10 uncontrolled-pH operation produced maximum SAP activity that was ca. 900 U cm −3 at t=21 h . According to the biomass and SAP production profiles, bioprocess was divided into two periods; Period I (0<t⩽10 h) covers the cell growth phase and Period II (10<t⩽24 h) covers the SAP production phase. In Period I in both operations, while the oxygen uptake rate (OUR) increased with the increase in initial pH the oxygen transfer rate (OTR) decreased. In Period II, among uncontrolled-pH operations OUR was the lowest at pH=7.10 while OTR decreased with the increase in initial pH. At the oxygen transfer condition applied, the bioprocess is biochemical reaction limited at all the conditions; nevertheless, with the decrease in initial pH, Damköhler number that is the oxygen transfer limitation decreases. Further, the perturbation effects of initial pH and the operation conditions on the intracellular reaction rates were calculated for Periods I and II by using the experimental data obtained and, the diversions in the pathways and certain metabolic reactions and potential strategies for improving SAP production are also discussed.

Principal component analysis of bioreactor fed-batch operation by computer simulation

Process control and principal component analysis of yeast fed-batch production is based on a computer model. The model is derived from intracellular reaction rates of key metabolic reactions during diauxillary growth, overall mass balances in reactor, and the PI regulation of the secondary product concentration. Principal components of the process matrix are evaluated by use of the QR algorithm. The results are discussed in view of process modelling, monitoring and adaptive control by a neural network model.

Effect of growth conditions on enzyme activities, intracellular kinetics and biomass yields of a new RuMP-type methylotroph (T15)

A new methylotrophic strain (T15), which employs the ribulose monophosphate (RuMP) cycle of formaldehyde assimilation, was isolated on the basis of high in vitro activities of formaldehyde and formate dehydrogenases (19 and 678 mU per mg protein, respectively). Serial subculturing of the strain in batch cultures, on 4 g/l CH3OH for 6 months, led to loss of substantial percentages of the NAD-linked formaldehyde (25%) and formate (98%) dehydrogenases. The activities of these two enzymes were partially recovered when cells were grown continuously at very low dilution rate (0.03 h−1). We found large variations (40 to 1000%) in the activities of other key enzymes of carbon-substrate oxidation (both linear and cyclic) and assimilation, in batch cultures with pure and mixed substrates, and in continuous cultures of different dilution rates. Key intracellular reaction rates, including those of the cyclic and linear substrate oxidation, were measured in vivo using a 14C-tracer technique in both continuous and batch cultures. The results indicate significant variations in these reaction rates, particularly those of linear and cyclic carbon oxidation. Overall, the cyclic oxidation appears to be employed to a larger (although not predominant) extent in strain T15 compared with another RuMP strain (L3) we have previously examined. T15 exhibits high biomass yields (up to 0.63 g cells per g CH3OH) and growth rates (up to 0.46 h−1) on CH3OH in batch cultures. CH3NH2 can also be utilized as a substrate. In continuous culture, T15 could be grown at dilution rates up to 0.36 h−1 with a corresponding biomass yield of 0.4. Examination of a large number of data on the biomass yields of strains T15 and L3 reveals that the large variations in yields derive from the variable branching of carbon flow between linear and cyclic oxidation and assimilation, rather than changes in the biosynthetic efficiency of carbon incorporation into biomass.

Intracellular reaction rates, enzyme activities and biomass yields in Methylomonas L3: growth rate and substrate composition effects

The effects of specific growth rate and medium feed composition on the metabolic reactions of methanol incorporation and oxidation have been studied in carbon-limited, chemostatic cultures of Methylomonas L3. An in situ radioisotopic tracer technique was employed. The in vivo rates of substrate-carbon flow and the corresponding steady-state levels of several key RuMP-type methylotrophic enzymes were determined over a range of dilution rates from 0.19 to 0.41 h-1 on methanol and methanol/formaldehyde substrates. It was determined that an absolute correlation does not exist between the in vivo specific carbon flux and the in vitro specific activity of any of the key enzymes studied. Oxidation of substrate-carbon via 6-phosphogluconate dehydrogenase is not stringently regulated in this methylotroph and the extent of its operation may be dependent on kinetic factors which make immediate cellular detoxification of formaldehyde imperative. As such, the cyclic oxidation mechanism in this methylotroph does not appear to be coupled to efficient energy utilization, since it was observed that high levels of the cyclic oxidation flux are commensurate with depressed biomass yields.

Red cell volume changes monitored using a new 31P NMR procedure

The application of NMR spectroscopy to whole cell systems has provided considerable insight into a range of cellular phenomena. 3’P NMR is used routinely for the estimation of intracellular pH (I, 2) and in assessing the energy status of whole cell samples (3). ‘H NMR has been used to monitor a variety of membrane transport processes (4) as well as a number of intracellular reactions (5). The rates of reactions inside the cell depend upon the concentrations of enzymes, substrates, and effector molecules and these, in turn, are determined by the intracellular volume. Changes in cell volume have, for example, been shown to affect the glycolytic rate in the human erythrocyte, mainly by altering the intracellular concentration of ATP, a regulator of glycolysis (6). Therefore, in any study of intracellular reaction rates, an understanding of how (and why) the cell volume may be changing during a particular experiment is important. We present here results obtained using a method whereby time-dependent changes in red cell volume may be monitored using “P NMR. The method is based on the observed cell volume dependence of the chemical shift of the “P NMR signal to dimethyl methylphosphonate. It is applicable to high hematocrit cell suspensions and was used to monitor the decrease in erythrocyte volume that followed the addition of the potassium ionophore, valinomycin, to cells suspended in a low-potassium medium. Erythrocyte suspensions were prepared from fresh blood obtained from the Red Cross Transfusion Service, New South Wales, Australia. The cells were washed twice in isotonic saline (0.154 M NaCl, 4°C) then three times in Krebs bicarbonate buffer (7) equilibrated beforehand with Carbogen (Oz/COz ; 19: 1) to give a pH of 7.3. All 3LP NMR spectra were recorded at 162 MHz using a Bruker WM 400 spectrometer operated in the Fourier transform mode at 37°C. Broadband proton decoupling was used throughout the experiments and the sample tubes were spun at - 16 Hz. Chemical shifts are quoted relative to that of 85% (v/v) phos- phoric acid. Dimethyl methylphosphonate (DMMP; CH3PO(OCH3)2; Aldrich, Mich.) is a small, neutral molecule, readily miscible with water. The “P NMR spectrum of DMMP in saline solution appeared as a single resonance (6 = 39.4 ppm). The addition of whole cells to the solution caused the signal to split into two. Furthermore, as the average red cell volume was altered by varying the extracellular