Respirometric 13C flux analysis--Part II: in vivo flux estimation of lysine-producing Corynebacterium glutamicum

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

Modeling and experimental design for metabolic flux analysis of lysine-producing Corynebacteria by mass spectrometry

Experimental design of (13)C-tracer studies for metabolic flux analysis with mass spectrometric determination of labeling patterns was performed for the central metabolism of Corynebacterium glutamicum comprising various flux scenarios. Ratio measurement of mass isotopomer pools of Corynebacterium products lysine, alanine, and trehalose is sufficient to quantify the flux partitioning ratios (i) between glycolysis and pentose phosphate pathways (Phi(PPP)), (ii) between the split pathways in the lysine biosynthesis (Phi(DH)), (iii) at the pyruvate node (Phi(PC)), and reversibilities of (iv) glucose 6-phosphate isomerase (zeta(PGI)), (v) at the pyruvate node (zeta(PC/PEPCK)), and (vi) of transaldolase and transketolases in the PPP. Weighted sensitivities for flux parameters were derived from partial derivatives to quantitatively evaluate experimental approaches and predict precision for estimated flux parameters. Deviation of intensity ratios from ideal values of 1 was used as weighting function. Weighted flux sensitivities can be used to identify optimal type and degree of tracer labeling or potential intensity ratios to be measured. Experimental design for lysine-producing strain C. glutamicum MH 20-22B (Marx et al., Biotechnol. Bioeng. 49, 111-129, 1996) and various potential mutants with different alterations in the flux pattern showed that specific tracer labelings are optimal to quantify a certain flux parameter uninfluenced by the overall flux situation. Identified substrates of choice are [1-(13)C]glucose for the estimation of Phi(PPP) and zeta(PGI) and a 1 : 1 mixture of [U-(12)C/U-(13)C]glucose for the determination of zeta(PC/PEPCK). Phi(PC) can be quantified by feeding [4-(13)C]glucose or [U-(12)C/U-(13)C]glucose (1 : 1), whereas Phi(DH) is accessible via [4-(13)C]glucose. The sensitivity for the quantification of a certain flux parameter can be influenced by superposition through other flux parameters in the network, but substrate and measured mass isotopomers of choice remain the same. In special cases, reduced labeling degree of the tracer substrate can increase the precision of flux analysis. Enhanced precision and flux information can be achieved via multiply labeled substrates. The presented approach can be applied for effective experimental design of (13)C tracer studies for metabolic flux analysis. Intensity ratios of other products such as glutamate, valine, phenylalanine, and riboflavin also sensitively reflect flux parameters, which underlines the great potential of mass spectrometry for flux analysis.     

Serial flux mapping of Corynebacterium glutamicum during fed-batch L-lysine production using the sensor reactor approach

Using our recently developed sensor reactor approach, lysine-producing, nongrowing Corynebacterium glutamicum MH20-22B cells were subjected to serial (13)C-labeling experiments for flux analysis during the leucine-limited fed-batch production phase in a 300-L bioreactor. Based on two-dimensional (2D) nuclear magnetic resonance (NMR) measurements of (13)C-labeling patterns of cytoplasmic free metabolites, metabolic flux distributions in the central metabolism were successfully determined. Focusing on the highly concentrated metabolite L-glutamate, the working hypothesis was validated that the equilibration of labeling patterns in intracellular pools was much faster (up to 9.45 min) than the labeling period (3 h) used in the experiments. Analysis of anaplerotic reactions revealed that highly selective lysine production was accompanied by a significant reduction of decarboxylating reactions from 10 mol% to only 2 mol%, whereas PEP/pyruvate-carboxylating fluxes remained constant at about 40 mol% of consumed glucose. These results support the conclusion that an optimized C. glutamicum L-lysine producer should possess increased PEP carboxylase and/or pyruvate carboxylase activity combined with downregulated, decarboxylating fluxes consuming oxaloacetate/malate. The findings also illustrate the usefulness of the sensor reactor approach in the study of industrial fermentations.     

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.     

Metabolic network analysis of lysine producing Corynebacterium glutamicum at a miniaturized scale

We present a straightforward approach comprising (13)C tracer experiments at 200-microL volume in 96-well microtiter plates with on-line measurement of dissolved oxygen for quantitative high-throughput metabolic network analysis at a miniaturized scale. This method was successfully applied for cultivation and (13)C metabolic flux analysis of two mutants of lysine producing Corynebacterium glutamicum (ATCC 13287 and ATCC 21543). Microtiter-plate cultivations showed excellent accordance in kinetics and stoichiometry of growth and product formation as well as in intracellular flux distributions as compared with parallel shake-flask experiments. These cultivations further allowed clear identification of strain-specific flux differences such as increased flux toward lysine, increased flux through the pentose phosphate pathway (PPP), decreased flux through the tricarboxylic (TCA) cycle, and increased dihydroxyacetone formation in C. glutamicum ATCC 21543 compared with ATCC 13287. The present approach has strong potential for broad quantitative screening of metabolic network activities, especially those involving high-cost tracer substrates.     

Metabolic fluxes in Corynebacterium glutamicum during lysine production with sucrose as carbon source

Metabolic fluxes in the central metabolism were determined for lysine-producing Corynebacterium glutamicum ATCC 21526 with sucrose as a carbon source, providing an insight into molasses-based industrial production processes with this organism. For this purpose, 13C metabolic flux analysis with parallel studies on [1-(13C)Fru]sucrose, [1-(13C)Glc]sucrose, and [13C6Fru]sucrose was carried out. C. glutamicum directed 27.4% of sucrose toward extracellular lysine. The strain exhibited a relatively high flux of 55.7% (normalized to an uptake flux of hexose units of 100%) through the pentose phosphate pathway (PPP). The glucose monomer of sucrose was completely channeled into the PPP. After transient efflux, the fructose residue was mainly taken up by the fructose-specific phosphotransferase system (PTS) and entered glycolysis at the level of fructose-1,6-bisphosphate. Glucose-6-phosphate isomerase operated in the gluconeogenetic direction from fructose-6-phosphate to glucose-6-phosphate and supplied additional carbon (7.2%) from the fructose part of the substrate toward the PPP. This involved supply of fructose-6-phosphate from the fructose part of sucrose either by PTS(Man) or by fructose-1,6-bisphosphatase. C. glutamicum further exhibited a high tricarboxylic acid (TCA) cycle flux of 78.2%. Isocitrate dehydrogenase therefore significantly contributed to the total NADPH supply of 190%. The demands for lysine (110%) and anabolism (32%) were lower than the supply, resulting in an apparent NADPH excess. The high TCA cycle flux and the significant secretion of dihydroxyacetone and glycerol display interesting targets to be approached by genetic engineers for optimization of the strain investigated.     

Determination of the fluxes in the central metabolism of Corynebacterium glutamicum by nuclear magnetic resonance spectroscopy combined with metabolite balancing

To determine the in vivo fluxes of the central metabolism we have developed a comprehensive approach exclusively based on the fundamental enzyme reactions known to be present, the fate of the carbon atoms of individual reactions, and the metabolite balance of the culture. No information on the energy balance is required, nor information on enzyme activities, or the directionalities of reactions. Our approach combines the power of (1)H-detected (13)C nuclear magnetic resonance spectroscopy to follow individual carbons with the simplicity of establishing carbon balances of bacterial cultures. We grew a lysine-producing strain of Corynebacterium glutamicum to the metabolic and isotopic steady state with [1-(13)C]glucose and determined the fractional enrichments in 27 carbon atoms of 11 amino acids isolated from the cell. Since precursor metabolites of the central metabolism are incorporated in an exactly defined manner in the carbon skeleton of amino acids, the fractional enrichments in carbons of precursor metabolites (oxaloacetate, glyceraldehyde 3-phosphate, erythrose 4-phosphate, etc.) became directly accessible. A concise and generally applicable mathematical model was established using matrix calculus to express all metabolite mass and carbon labeling balances. An appropriate all-purpose software for the iterative solution of the equations is supplied. Applying this comprehensive methodology to C. glutamicum, all major fluxes within the central metabolism were determined. The result is that the flux through the pentose phosphate pathway is 66.4% (relative to the glucose input flux of 1.49 mmol/g dry weight h), that of entry into the tricarboxylic acid cycle 62.2%, and the contribution of the succinylase pathway of lysine synthesis 13.7%. Due to the large amount and high quality of measured data in vivo exchange reactions could also be quantitated with particularly high exchange rates within the pentose phosphate pathway for the ribose 5-phosphate transketolase reaction. Moreover, the total net flux of the anaplerotic reactions was quantitated as 38.0%. Most importantly, we found that in vivo one component within these anaplerotic reactions is a back flux from the carbon 4 units of the tricarboxylic acid cycle to the carbon 3 units of glycolysis of 30.6%. (c) 1996 John Wiley & Sons, Inc.     

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.     

Comparative metabolic flux analysis of lysine-producing Corynebacterium glutamicum cultured on glucose or fructose

A comprehensive approach to (13)C tracer studies, labeling measurements by gas chromatography-mass spectrometry, metabolite balancing, and isotopomer modeling, was applied for comparative metabolic network analysis of lysine-producing Corynebacterium glutamicum on glucose or fructose. Significantly reduced yields of lysine and biomass and enhanced formation of dihydroxyacetone, glycerol, and lactate in comparison to those for glucose resulted on fructose. Metabolic flux analysis revealed drastic differences in intracellular flux depending on the carbon source applied. On fructose, flux through the pentose phosphate pathway (PPP) was only 14.4% of the total substrate uptake flux and therefore markedly decreased compared to that for glucose (62.0%). This result is due mainly to (i) the predominance of phosphoenolpyruvate-dependent phosphotransferase systems for fructose uptake (PTS(Fructose)) (92.3%), resulting in a major entry of fructose via fructose 1,6-bisphosphate, and (ii) the inactivity of fructose 1,6-bisphosphatase (0.0%). The uptake of fructose during flux via PTS(Mannose) was only 7.7%. In glucose-grown cells, the flux through pyruvate dehydrogenase (70.9%) was much less than that in fructose-grown cells (95.2%). Accordingly, flux through the tricarboxylic acid cycle was decreased on glucose. Normalized to that for glucose uptake, the supply of NADPH during flux was only 112.4% on fructose compared to 176.9% on glucose, which might explain the substantially lower lysine yield of C. glutamicum on fructose. Balancing NADPH levels even revealed an apparent deficiency of NADPH on fructose, which is probably overcome by in vivo activity of malic enzyme. Based on these results, potential targets could be identified for optimization of lysine production by C. glutamicum on fructose, involving (i) modification of flux through the two PTS for fructose uptake, (ii) amplification of fructose 1,6-bisphosphatase to increase flux through the PPP, and (iii) knockout of a not-yet-annotated gene encoding dihydroxyacetone phosphatase or kinase activity to suppress overflow metabolism. Statistical evaluation revealed high precision of the estimates of flux, so the observed differences for metabolic flux are clearly substrate specific.     

13C metabolic flux analysis for larger scale cultivation using gas chromatography-combustion-isotope ratio mass spectrometry

¹³C-based metabolic flux analysis (¹³CMFA) is limited to smaller scale experiments due to very high costs of labeled substrates. We measured ¹³C enrichment in proteinogenic amino acid hydrolyzates using gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) from a series of parallel batch cultivations of Corynebacterium glutamicum utilizing mixtures of natural glucose and [1-¹³C] glucose, containing 0%, 0.5%, 1%, 2%, and 10% [1-¹³C] glucose. Decreasing the [1-¹³C] glucose content, kinetic isotope effects played an increasing role but could be corrected. From the corrected ¹³C enrichments in vivo fluxes in the central metabolism were determined by numerical optimization. The obtained flux distribution was very similar to those obtained from parallel labeling experiments using conventional high labeling GC-MS method and to published results. The GC-C-IRMS-based method involving low labeling degree of expensive tracer substrate, e.g. 1%, is well suited for larger laboratory and industrial pilot scale fermentations.     

Amplified expression of fructose 1,6-bisphosphatase in Corynebacterium glutamicum increases in vivo flux through the pentose phosphate pathway and lysine production on different carbon sources

The overexpression of fructose 1,6-bisphosphatase (FBPase) in Corynebacterium glutamicum leads to significant improvement of lysine production on different sugars. Amplified expression of FBPase via the promoter of the gene encoding elongation factor TU (EFTU) increased the lysine yield in the feedback-deregulated lysine-producing strain C. glutamicum lysCfbr by 40% on glucose and 30% on fructose or sucrose. Additionally formation of the by-products glycerol and dihydroxyacetone was significantly reduced in the PEFTUfbp mutant. As revealed by 13C metabolic flux analysis on glucose the overexpression of FBPase causes a redirection of carbon flux from glycolysis toward the pentose phosphate pathway (PPP) and thus leads to increased NADPH supply. Normalized to an uptake flux of glucose of 100%, the relative flux into the PPP was 56% for C. glutamicum lysCfbr PEFTUfbp and 46% for C. glutamicum lysCfbr. The flux for NADPH supply was 180% in the PEFTUfbp strain and only 146% in the parent strain. Amplification of FBPase increases the production of lysine via an increased supply of NADPH. Comparative studies with another mutant containing the sod promoter upstream of the fbp gene indicate that the expression level of FBPase relates to the extent of the metabolic effects. The overexpression of FBPase seems useful for starch- and molasses-based industrial lysine production with C. glutamicum. The redirection of flux toward the PPP should also be interesting for the production of other NADPH-demanding compounds as well as for products directly stemming from the PPP.     

COMPLETE-MFA: Complementary parallel labeling experiments technique for metabolic flux analysis

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

Respirometric 13C flux analysis, Part I: design, construction and validation of a novel multiple reactor system using on-line membrane inlet mass spectrometry

A novel method for (13)C flux analysis based on on-line CO(2) labeling measurements is presented. This so-called respirometric (13)C flux analysis requires multiple parallel (13)C labeling experiments using differently labeled tracer substrates. In Part I of the work, a membrane-inlet mass spectrometry-based measurement system with 6 parallel reactors with each 12 ml liquid volume and associated experimental and computational methods for the respirometric (13)C data acquisition and evaluation are described. Signal dynamics after switching between membrane probes follow exactly first-order allowing extrapolation to steady state. Each measurement cycle involving 3 reactors takes about 2 min. After development of a dynamic calibration method, the suitability and reliability of the analysis was examined with a lysine-producing mutant of Corynebacterium glutamicum using [1-(13)C(1)], [6-(13)C(1)], [1,6-(13)C(2)] glucose. Specific rates of oxygen uptake and CO(2) production were estimated with an error less than +/-0.3 mmol g(-1) h(-1) and had +/-3% to +/-10% deviations between parallel reactors which is primarily caused by inaccuracies in initial biomass concentration. The respiratory quotient could be determined with an uncertainty less than +/-0.02 and varied only +/-3% between reactors. Fractional labeling of CO(2) was estimated with much higher precision of about +/-0.001 to +/-0.005. The detailed statistical analysis suggested that these data should be of sufficient quality to allow physiological interpretation and metabolic flux estimation. The obtained data were applied for the respirometric (13)C metabolic flux analysis in Part II.     

Response of the central metabolism in Corynebacterium glutamicum to the use of an NADH-dependent glutamate dehydrogenase

The extensive use of 13C enrichments in precursor metabolites for flux quantification does not rely on NADPH stoichiometries and can therefore be used to quantify reducing power fluxes. As an application of this concept, the NADPH fluxes were quantified in an L-lysine producer of Corynebacterium glutamicum grown into metabolic and isotopic steady state with [1-13C]glucose. In this case, where the organism's NADPH-dependent glutamate dehydrogenase consumes reducing power, the NADPH flux generated is 210% (molar flux relative to glucose uptake rate) with its major part (72% of the total) generated via the pentose phosphate pathway activity. An isogenic strain in which the glutamate dehydrogenase of C. glutamicum was replaced by the NADH-dependent glutamate dehydrogenase of Peptostreptococcus asaccharolyticus was made and the metabolite fluxes were again estimated. The major response to this local perturbation is a drastically reduced NADPH generation of only 139%. Most of the NADPH (62% of the total) is now generated via the tricarboxylic acid cycle activity. This shows the extraordinary flexibility of the central metabolism and provides a picture of the global regulatory properties of the central metabolism. Furthermore, a detailed analysis of the fluxes and exchange fluxes within the anaplerotic reactions is given. It is hypothesized that these reactions might also serve to balance the total reducing power budget as well as the energy budget within the cell.     

Bidirectional reaction steps in metabolic networks: IV. Optimal design of isotopomer labeling experiments

This article generalizes the statistical tools for the evaluation of carbon-labeling experiments that have been developed for the case of positional enrichment systems in part II of this series to the general case of isotopomer systems. For this purpose, a new generalized measurement equation is introduced that can describe all kinds of measured data, like positional enrichments, relative (13)C nuclear magnetic resonance ((13)C NMR) multiplet intensities, or mass isotopomer fractions produced with mass spectroscopy (MS) instruments. Then, to facilitate the specification of the various measurement procedures available, a new flexible textual notation is introduced from which the complicated generalized measurement equations are generated automatically. Based on these measurement equations, a statistically optimal flux estimator is established and parameter covariance matrices for the flux estimation are computed. Having implemented these tools, different kinds of labeling experiments can be compared by using statistical quality measures. A general framework for the optimal design of carbon-labeling experiments is established on the basis of this method. As an example it is applied to the Corynebacterium network from part II extended by various NMR and MS measurements. In particular, the positional enrichment, multiplet, or mass isotopomer measurements with the greatest information content for flux estimation are computed (measurement design) and various differently labeled input substrates are compared with respect to flux estimation (input design). It is examined in detail how the measurement procedure influences the estimation quality of specific fluxes like the pentose phosphate pathway influx.     

A flux model of glycolysis and the oxidative pentosephosphate pathway in developing Brassica napus embryos

Developing oilseeds synthesize large quantities of triacylglycerol from sucrose and hexose. To understand the fluxes involved in this conversion, a quantitative metabolic flux model was developed and tested for the reaction network of glycolysis and the oxidative pentose phosphate pathway (OPPP). Developing Brassica napus embryos were cultured with [U-13C6]glucose, [1-13C]glucose, [6-13C]glucose, [U-13C12]sucrose, and/or [1,2-13C2]glucose and the labeling patterns in amino acids, lipids, sucrose, and starch were measured by gas chromatography/mass spectrometry and NMR. Data were used to verify a reaction network of central carbon metabolism distributed between the cytosol and plastid. Computer simulation of the steady state distribution of isotopomers in intermediates of the glycolysis/OPPP network was used to fit metabolic flux parameters to the experimental data. The observed distribution of label in cytosolic and plastidic metabolites indicated that key intermediates of glycolysis and OPPP have similar labeling in these two compartments, suggesting rapid exchange of metabolites between these compartments compared with net fluxes into end products. Cycling between hexose phosphate and triose phosphate and reversible transketolase velocity were similar to net glycolytic flux, whereas reversible transaldolase velocity was minimal. Flux parameters were overdetermined by analyzing labeling in different metabolites and by using data from different labeling experiments, which increased the reliability of the findings. Net flux of glucose through the OPPP accounts for close to 10% of the total hexose influx into the embryo. Therefore, the reductant produced by the OPPP accounts for at most 44% of the NADPH and 22% of total reductant needed for fatty acid synthesis.