Bioreaction Network Research Articles

Metabolic flux analysis of CHO cells in perfusion culture by metabolite balancing and 2D [ 13C, 1H] COSY NMR spectroscopy

The physiological state of CHO cells in perfusion culture was quantified by determining fluxes through the bioreaction network using 13C glucose and 2D-NMR spectroscopy. CHO cells were cultivated in a 2.5 L perfusion bioreactor with glucose and glutamine as the primary carbon and energy sources. The reactor was inoculated at a cell density of 8×10 6 cells/mL and operated at ∼10×10 6 cells/mL using unlabeled glucose for the first 13 days. The second phase lasted 12 days and the medium consisted of 10% [U- 13C]glucose, 40% labeled [1- 13C]glucose with the balance unlabeled. After the culture attained isotopic steady state, biomass samples from the last 3 days of cultivation were considered representative and used for flux estimation. They were hydrolyzed and analyzed by 2D [ 13C, 1H] COSY measurements using the heteronuclear single quantum correlation sequence with gradients for artifacts suppression. Metabolic fluxes were determined using the 13C-Flux software package by minimizing the residuals between the experimental and the simulated NMR data. Normalized residuals exhibited a Gaussian distribution indicating good model fit to experimental data. The glucose consumption rate was 5-fold higher than that of glutamine with 41% of glucose channeled through the pentose phosphate pathway. The fluxes at the pyruvate branch point were almost equally distributed between lactate and the TCA cycle (55% and 45%, respectively). The anaplerotic conversion of pyruvate to oxaloacetate by pyruvate carboxylase accounted for 10% of the pyruvate flux with the remaining 90% entering the TCA cycle through acetyl-CoA. The conversion of malate to pyruvate catalyzed by the malic enzyme was 70% higher than that for the anaplerotic reaction catalyzed by pyruvate carboxylase. Most amino acid catabolic and biosynthetic fluxes were significantly lower than the glycolytic and TCA cycle fluxes. Metabolic flux data from NMR analysis validated a simplified model where metabolite balancing was used for flux estimation. In this reduced flux space, estimates from these two methods were in good agreement. This simplified model can routinely be used in bioprocess development experiments to estimate metabolic fluxes with much reduced analytical investment. The high resolution flux information from 2D-NMR spectroscopy coupled with the capability to validate a simplified metabolite balancing based model for routine use make 13C-isotopomer analysis an attractive bioprocess development tool for mammalian cell cultures.

Bioreaction network flux analysis for human protein producing Bacillus subtilis based on genome-scale model

To interpret the effect of human protein gene, e.g., human growth hormone gene hGH, on the intracellular bioreaction network of Bacillus subtilis, the intracellular reaction fluxes were calculated by solving the mass-balance-based genome-scale mathematical model, at pseudo-steady state by using bioreactor data. The bioreaction network consists of 1340 reactions including 990 metabolites. Reaction fluxes in B. subtilis carrying pMK4 ::pre(subC)::hGH (rBsHGH) and B. subtilis carrying merely pMK4 (rBsP) were calculated to identify the existence of pathways and to elucidate the physiological state of the cells. Considering the characteristic cell and recombinant hGH (rhGH) concentration profiles of rBsHGH, the bioprocess was divided into five periods. The reaction network of rBsHGH started operation with 328 reactions (Period I: 0< t<4 h) and lowered to 313 in Period II (4< t<12 h), increased to 314 in Period III (12< t<16 h) by starting rhGH synthesis, and decreased to 296 reactions in Period IV (16< t<24 h) where cell and rhGH formation proceeded; further, decreased to 135 reactions in Period V (24< t<32 h) where cell formation ends and rhGH synthesis continues. However, the reaction network of rBsP started operation with 322 reactions, and lowered to 318 in Period II, and decreased to 43 reactions in Period III where cell formation ends. The flux of the reaction R1340 toward rhGH was the highest (2.0×10 −4 mmol g −1DW h −1) in Period V. The foreign protein gene stimulated and prolonged the cell synthesis (R1339) till the end of Period IV. To increase rhGH production further an exponential glucose feeding strategy between Periods III–V to maintain the specific growth rate μ within 0.02–0.08 h −1, is proposed.

Systematic quantification of complex metabolic flux networks using stable isotopes and mass spectrometry.

Metabolic fluxes provide a detailed metric of the cellular metabolic phenotype. Fluxes are estimated indirectly from available measurements and various methods have been developed for this purpose. Of particular interest are methods making use of stable isotopic tracers as they enable the estimation of fluxes at a high resolution. In this paper, we present data validating the use of mass spectrometry (MS) for the quantification of complex metabolic flux networks. In the context of the lysine biosynthesis flux network of Corynebacterium glutamicum (ATCC 21799) under glucose limitation in continuous culture, operating at 0.1 x h(-1) after the introduction of 50% [1-13C]glucose, we deploy a bioreaction network analysis methodology for flux determination from mass isotopomer measurements of biomass hydrolysates, while thoroughly addressing the issues of measurement accuracy, flux observability and data reconciliation. The analysis enabled the resolution of the involved anaplerotic activity of the microorganism using only one labeled substrate, the determination of the range of most of the exchange fluxes and the validation of the flux estimates through satisfaction of redundancies. Specifically, we determined that phosphoenolpyruvate carboxykinase and synthase do not carry flux at these experimental conditions and identified a high futile cycle between oxaloacetate and pyruvate, indicating a highly active in vivo oxaloacetate decarboxylase. Both results validated previous in vitro activity measurements. The flux estimates obtained passed the chi2 statistical test. This is a very important result considering that prior flux analyses of extensive metabolic networks from isotopic measurements have failed criteria of statistical consistency.

Metabolic flux analysis of halogenated monoterpene biosynthesis in microplantlets of the macrophytic red alga Ochtodes secundiramea

The bioprocess engineering of marine macroalgae (i.e. seaweeds) for the production of secondary metabolites is an emerging area of marine biotechnology. One novel system is the biosynthesis of halogenated monoterpenes by “microplantlet” suspension cultures derived from the red alga Ochtodes secundiramea. This biosynthetic platform has three principal components: elaboration of myrcene from geranyl diphosphate (GPP); bromonium-ion promoted halogenation of myrcene to 10 E-bromomyrcene, 3-chloro-10 E-bromo-α-myrcene, and 3,10 E-dibromomyrcene; bromonium-ion promoted cyclization of myrcene to Apakaochtodene B. In this study, a metabolic flux analysis on halogenated monoterpene biosynthesis was performed. To facilitate this effort, a “bromine free” cell line of O. secundiramea microplantlets was developed where biohalogenation was temporarily disabled but myrcene biosynthesis was still enabled. This cell line was cultivated within an airlift photobioreactor under nutrient medium perfusion. Halogenated monoterpene biosynthesis was “turned on” by coordinated addition of bromide and vanadate (a co-factor for vanadium bromoperoxidase) to the perfusion medium. From these experiments, the effects of bromide and vanadate delivery on the metabolic flux of each metabolite were determined. Bromination of myrcene at its Δ 6–10 olefinic bond was the dominant branch of the bioreaction network, whereas chlorination steps in the pathway were “weakly rigid”. This study represents the first application of metabolic engineering principles to the analysis and manipulation of secondary metabolism in macrophytic marine organisms.

Overexpression of a serine alkaline protease gene in Bacillus licheniformis and its impact on the metabolic reaction network

This work reports on cloning of serine alkaline protease (SAP) encoding gene subC to a multi-copy plasmid and its expression in Bacillus licheniformis with the quantitative impact of overexpression of the subC gene on metabolic flux distributions. Bioprocess characteristics of the wild-type and the recombinant B. licheniformis were investigated in a defined simple synthetic medium with glucose as the sole carbon source under well-defined bioreactor-operation conditions. Significant physiological changes were observed in the recombinant B. licheniformis in response to altered bioreactor-operation conditions, i.e. initial glucose concentration. The growth kinetics of microbial cells were investigated prior to the investigation of intracellular reactions and rates within the cell; the unstructured substrate inhibition and Monod models were found valid for the wild-type and recombinant B. licheniformis, respectively. Optimum initial glucose concentration for maximum SAP production and the corresponding cultivation time of the recombinant B. licheniformis shifted respectively from C G0=6 to 8 kg m −3 and from t=43 to 67 h. The maximum SAP activity was obtained as 950 U cm −3 with the recombinant B. licheniformis, which was ca. 2.5-fold higher than that of the wild-type. Carbon fluxes through the central metabolic pathways in the wild-type and recombinant B. licheniformis were calculated, using a mass balance-based mathematical model that contains 105 metabolites and 148 reaction fluxes and the time profiles of glucose, dry cell weight, organic acids, amino acids and SAP obtained in 3.5 dm 3 bioreactor systems at C G0=6 kg m −3 for the exponential growth phase and the SAP production phase. The bioreaction network flux analyses were first accomplished by using the theoretical data-based approach; and then by using the theoretical data-based capacity analysis approach. During the SAP synthesis period, the actual fluxes of the glycolysis pathway, the pentose phosphate pathway, the tricarboxylic acid cycle, the amino acids biosynthetic pathways (and, consequently, SAP synthesis) are higher in the recombinant B. licheniformis strain than in the wild-type. Further, the normalised relative flux values of all the pathways, except the glycolysis pathway, change considerably in the recombinant bacteria. The effectiveness factor, defined as the SAP synthesis rate per maximum possible SAP synthesis rate was η=0.20 for the recombinant B. licheniformis. This indicates the possibility of a further increase in SAP production through metabolic engineering, and potential strategies to achieve this are also discussed.

Metabolic engineering of indene bioconversion in Rhodococcus sp.

We have applied the methodology of metabolic engineering in the investigation of the enzymatic bioreaction network in Rhodococcus sp. that catalyzes the bioconversion of indene to (2R)-indandiol suitable for the synthesis of cis-1-amino-2-indanol, a precursor of the HIV protease inhibitor, Crixivan. A chemostat with a novel indene air delivery system was developed to facilitate the study of steady state physiology of Rhodococcus sp. 124. Prolonged cultivation of this organism in a continuous flow system led to the evolution of a mutant strain, designated KY1, with improved bioconversion properties, in particular a twofold increase in yield of (2R)-indandiol relative to 124. Induction studies with both strains indicated that KY1 lacked a toluene-inducible dioxygenase activity present in 124 and responsible for the formation of undesired byproducts. Flux analysis of indene bioconversion in KY1 performed using steady state metabolite balancing and labeling with [14C]-tracers revealed that at least 94% of the indene is oxidized by a monooxygenase to indan oxide that is subsequently hydrolyzed to trans-(1R,2R)-indandiol and cis-(1S,2R)-indandiol. This analysis identified several targets in KY1 for increasing (2R)-indandiol product yield. Most promising among them is the selective hydrolysis of indan oxide to trans-(1R,2R)-indandiol through expression of an epoxide hydrolase or modification of culture conditions.

Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction

The two main contributions of this article are the solidification of Corynebacterium glutamicum biochemistry guided by bioreaction network analysis, and the determination of basal metabolic flux distributions during growth and lysine synthesis. Employed methodology makes use of stoichiometrically based mass balances to determine flux distributions in the C. glutamicum metabolic network. Presented are a brief description of the methodology, a thorough literature review of glutamic acid bacteria biochemistry, and specific results obtained through a combination of fermentation studies and analysis-directed intracellular assays. The latter include the findings of the lack of activity of glyoxylate shunt, and that phosphoenolpyruvate carboxylase (PPC) is the only anaplerotic reaction expressed in C. glutamicum cultivated on glucose minimal media. Network simplifications afforded by the above findings facilitated the determination of metabolic flux distributions under a variety of culture conditions and led to the following conclusions. Both the pentose phosphate pathway and PPC support significant fluxes during growth and lysine overproduction, and that flux partitioning at the glucosa-6-phosphate branch point does not appear to limit lysine synthesis.

Metabolic flux distributions inCorynebacterium glutamicum during growth and lysine overproduction

The two main contributions of this article are the solidification of Corynebacterium glutamicum biochemistry guided by bioreaction network analysis, and the determination of basal metabolic flux distributions during growth and lysine synthesis. Employed methodology makes use of stoichiometrically based mass balances to determine flux distributions in the C. glutamicum metabolic network. Presented are a brief description of the methodology, a thorough literature review of glutamic acid bacteria biochemistry, and specific results obtained through a combination of fermentation studies and analysis-directed intracellular assays. The latter include the findings of the lack of activity of glyoxylate shunt, and that phosphoenolpyruvate carboxylase (PPC) is the only anaplerotic reaction expressed in C. glutamicum cultivated on glucose minimal media. Network simplifications afforded by the above findings facilitated the determination of metabolic flux distributions under a variety of culture conditions and led to the following conclusions. Both the pentose phosphate pathway and PPC support significant fluxes during growth and lysine overproduction, and that flux partitioning at the glucosa-6-phosphate branch point does not appear to limit lysine synthesis. © 1993 John Wiley & Sons, Inc.

Bioreaction Network Topology and Metabolic Flux Ratio Analysis by Biosynthetic Fractional 13C Labeling and Two-Dimensional NMR Spectroscopy

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.

Analysis of the pathway structure of metabolic networks

Metabolic networks comprise a multitude of enzymatic reactions carrying out various functions related to cell growth and product formation. Although such reactions are occasionally organized into biochemical pathways, a formal procedure is desired to identify the independent pathways in a bioreaction network and the degree of engagement of each individual reaction in these pathways. We present a procedure for the identification of the independent pathways of bioreaction networks of any size and complexity. The method makes use of the steady-state internal metabolite stoichiometry matrix and defines the independent pathways through the reaction membership of its kernel matrix. Examples from the aromatic amino acid biosynthetic pathway and central carbon metabolism of cells in culture are provided to illustrate the method. Applications to the analysis of the control structure of bioreaction networks are also discussed.

Metabolic flux distributions in Corynebacterium glutamicum during growth and lysine overproduction

The two main contributions of this are the solidification of Corynebacterium glutamicum biochemistry guided by bioreaction network analysis, and the determination of bansal metabolic flux distributions during growth and lysine synthesis. Employed methodology makes use of stoichiometrically based mass balances to determine flux distributions in the C. glutamicum metabolic network. Presented are a brief description of the methodology, a through literature review of glutamic acid bacteria biochemistry, and specific results obtained through a combination of fermentation studies and analysis-directed intracellular assays. The latter include the findings of the lack of activity of glyoxylate shunt, and that phosphoenolpyruvate carboxylase (PPC) is the only anaplerotic reaction expressed in C. glutamicum cultivated on glucose minimal media. Network simplifications afforded by the above findings facilitated the determination of metabolic flux distributions under a variety of culture conditions and led to the following conclusions. Both the pentose phosphate pathway and PPC support fluxes during growth and lysine overproduction branch point does not appear to limit lysine synthesis.