Phenotypic characterization of <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> using elementary modes towards synthesis of amino acids

Elementary flux mode (EFM) analysis is a powerful tool to represent the metabolic network structure and can be further utilized for flux analysis. The method enables characterization and quantification of feasible phenotypes in microbes. EFM analysis was employed to characterize the phenotype of Corynebacterium glutamicum to yield various amino acids. The metabolic network of C. glutamicum yielded 62 elementary modes by incorporating the accumulation of amino acids namely, lysine, alanine, valine, glutamine and glutamate. The analysis also allowed us to compute the maximum theoretical yield for the synthesis of various amino acids. These 62 elementary modes were further used to obtain optimal phenotypic space towards accumulation of biomass and lysine. The study indicated that the optimal solution space from 62 elementary modes forms a super space which incorporates various mutants including lysine producing strain of C. glutamicum. The analysis was also extended to obtain sensitivity of the network to variation in the stoichiometry of NADP in the definition of biomass.     

Lysine overproducing Corynebacterium glutamicum is characterized by a robust linear combination of two optimal phenotypic states

A homoserine auxotroph strain of Corynebacterium glutamicum accumulates storage compound trehalose with lysine when limited by growth. Industrially lysine is produced from C. glutamicum through aspartate biosynthetic pathway, where enzymatic activity of aspartate kinase is allosterically controlled by the concerted feedback inhibition of threonine plus lysine. Ample threonine in the medium supports growth and inhibits lysine production (phenotype-I) and its complete absence leads to inhibition of growth in addition to accumulating lysine and trehalose (phenotype-II). In this work, we demonstrate that as threonine concentration becomes limiting, metabolic state of the cell shifts from maximizing growth (phenotype-I) to maximizing trehalose phenotype (phenotype-II) in a highly sensitive manner (with a Hill coefficient of 4). Trehalose formation was linked to lysine production through stoichiometry of the network. The study demonstrated that the net flux of the population was a linear combination of the two optimal phenotypic states, requiring only two experimental measurements to evaluate the flux distribution. The property of linear combination of two extreme phenotypes was robust for various medium conditions including varying batch time, initial glucose concentrations and medium osmolality.     

Osmotic stress response in <Emphasis Type="Italic">C</Emphasis>. <Emphasis Type="Italic">glutamicum</Emphasis>: impact of channel- and transporter-mediated potassium accumulation

Potassium accumulation is an essential aspect of bacterial response to diverse stress situations; consequently its uptake plays a pivotal role. Here, we show that the Gram-positive soil bacterium Corynebacterium glutamicum which is employed for the large-scale industrial production of amino acids requires potassium under conditions of ionic and non-ionic osmotic stress. Besides the accumulation of high concentrations of potassium contributing significantly to the osmotic potential of the cytoplasm, we demonstrate that glutamate is not the counter ion for potassium under these conditions. Interestingly, potassium is required for the activation of osmotic stress-dependent expression of the genes betP and proP. The Kup-type potassium transport system which is present in C. glutamicum in addition to the potassium channel CglK does not contribute to potassium uptake at conditions of hyperosmotic stress. Furthermore, we established a secondary carrier of the KtrAB type from C. jeikeium in C. glutamicum thus providing an experimental comparison of channel- and carrier-mediated potassium uptake under osmotic stress. While at low potassium availability, the presence of the KtrAB transporter improves both potassium accumulation and growth of C. glutamicum upon osmotic stress, at proper potassium supply, the channel CglK is sufficient.     

Metabolic responses to pyruvate kinase deletion in lysine producing Corynebacterium glutamicum

Background

Pyruvate kinase is an important element in flux control of the intermediate metabolism. It catalyzes the irreversible conversion of phosphoenolpyruvate into pyruvate and is under allosteric control. In Corynebacterium glutamicum, this enzyme was regarded as promising target for improved production of lysine, one of the major amino acids in animal nutrition. In pyruvate kinase deficient strains the required equimolar ratio of the two lysine precursors oxaloacetate and pyruvate can be achieved through concerted action of the phosphotransferase system (PTS) and phosphoenolpyruvate carboxylase (PEPC), whereby a reduced amount of carbon may be lost as CO₂ due to reduced flux into the tricarboxylic acid (TCA) cycle. In previous studies, deletion of pyruvate kinase in lysine-producing C. glutamicum, however, did not yield a clear picture and the exact metabolic consequences are not fully understood.

Results

In this work, deletion of the pyk gene, encoding pyruvate kinase, was carried out in the lysine-producing strain C. glutamicum lysC^fbr, expressing a feedback resistant aspartokinase, to investigate the cellular response to deletion of this central glycolytic enzyme. Pyk deletion was achieved by allelic replacement, verified by PCR analysis and the lack of in vitro enzyme activity. The deletion mutant showed an overall growth behavior (specific growth rate, glucose uptake rate, biomass yield) which was very similar to that of the parent strain, but differed in slightly reduced lysine formation, increased formation of the overflow metabolites dihydroxyacetone and glycerol and in metabolic fluxes around the pyruvate node. The latter involved a flux shift from pyruvate carboxylase (PC) to PEPC, by which the cell maintained anaplerotic supply of the TCA cycle. This created a metabolic by-pass from PEP to pyruvate via malic enzyme demonstrating its contribution to metabolic flexibility of C. glutamicum on glucose.

Conclusion

The metabolic flux analysis performed illustrates the high flexibility of the metabolic network of C. glutamicum to compensate for external perturbation. The organism could almost maintain its growth and production performance through a local redirection of the metabolic flux, thereby fulfilling all anabolic and catabolic needs. The formation of the undesired overflow metabolites dihydroxyacetone and glycerol, in the deletion mutant, however, indicates a limiting capacity of the metabolism down-stream of their common precursor glyceraldehyde 3-phosphate and opens possibilities for further strain engineering.     

Growth of Corynebacterium glutamicum in ammonium- and potassium-limited continuous cultures under high osmotic pressure

 In order to determine the possible effect of nutrient limitations on the response of Corynebacterium glutamicum to a saline osmotic up-shock, the bacteria were grown in continuous cultures, at osmotic pressures of 0.4 osmol/kg and 1.2 osmol/kg, under ammonia and potassium limitation. At the low osmolality of 0.4 osmol/kg, the glutamate and proline levels of 15 mg/g and 5 mg/g dry weight respectively were lower than previously reported in glucose-limited continuous cultures (50 mg/g and 10 mg/g dry weight respectively). On the other hand, the internal trehalose pool was much higher at 40 mg/g dry weight. When the medium osmolality was increased to 1.2 osmol/kg by NaCl addition, under ammonia limitation, the proline content rose from 5 mg/g to 20 mg/g dry weight and the trehalose content from 40 mg/g to 70 mg/g dry weight, whereas the intracellular pool of glutamate remained essentially constant. An increase in the internal sodium content was also observed. Similar results were found for the internal pool of glutamate, proline and trehalose when C. glutamicum was grown under potassium limitations at an osmolality of 1.2 osmol/kg. There were also higher levels of sodium ions, glutamine and alanine. According to the present results, whereas proline was previously reported to be the dominantly accumulated osmoprotectant in C. glutamicum grown under glucose limitations, under ammonia and potassium limitations trehalose represented the dominantly synthesized metabolite. Received: 19 December 1995/Received revision: 9 April 1996/Accepted: 15 April 1996     

Metabolic flux redistribution in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> in response to osmotic stress

Osmotic stress constitutes a major bacterial stress factor in the soil and during industrial fermentation. In this paper, we quantified the metabolic response, in terms of metabolic flux redistribution, of a lysine-overproducing strain of Corynebacterium glutamicum grown under continuous culture, to gradually increasing osmolality. Oxygen and carbon dioxide evolution rates, and the changes in concentration of extracellular, as well as intracellular, metabolites were measured throughout the osmotic gradient. The metabolic fluxes were estimated from these measurements and from the mass balance constraints at each metabolite-node of the assumed metabolic reaction network. Our results show that formation rates of compatible solutes--trehalose first and proline at a later stage of the gradient--increased with osmotic stress to equilibrate the external osmotic pressure. Estimated flux distributions indicate that the observed increase in the glucose specific uptake rate with osmotic stress is channeled through the main energy generating pathways-- glycolysis and the tricarboxylic acid cycle--while the flux through the pentose phosphate pathway remains constant throughout the gradient. This results in a significant increase in the net specific ATP production rate, which may possibly be used to support the higher energy requirements required for cellular maintenance at high osmolalities. Finally, nodal analysis confirmed that the PEP/pyruvate node is essentially rigid and that the glucose-6-phosphate, oxaloacetate and alpha-ketoglutarate nodes are flexible and therefore adaptable to changes in osmotic pressure in C. glutamicum.     

Changes in intracellular composition in response to hyperosmotic stress of NaCl, sucrose or glutamic acid in Brevibacterium lactofermentum and Corynebacterium glutamicum

Changes in intracellular composition after hyperosmotic shock were studied in the lysine-producing mutant Brevibacterium lactofermentum NRRL B-11470 and the wild-type Corynebacterium glutamicum ATCC 13032. Both strains accumulated betaine, proline, glutamic acid, glutamine and trehalose in response to stress. The accumulated amino acids were synthesized by the cells, while betaine and trehalose were taken up from the medium. The contribution of synthesized osmoregulators was highest in C. glutamicum. In a sucrose-limited continuous culture, the increased outer osmotic pressure was balanced within 15 min for C. glutamicum and somewhat later in B. lactofermentum. The rapid regulation was due to both accumulation of osmoregulators, and shrinkage of cell and cytoplasmic volume. Immediately after shock, glutamine and glutamic acid were the dominating osmolytes. During the adaptation process, glutamine was replaced by the better osmoprotectant proline. In betaine-enriched cultures, betaine accumulation increased at the expense of glutamic acid, glutamine and trehalose. The total intracellular concentration of osmolytes increased linearly with increasing stress for all stress factors.     

Analysis of optimal phenotypic space using elementary modes as applied to Corynebacterium glutamicum

Background  

Quantification of the metabolic network of an organism offers insights into possible ways of developing mutant strain for better productivity of an extracellular metabolite. The first step in this quantification is the enumeration of stoichiometries of all reactions occurring in a metabolic network. The structural details of the network in combination with experimentally observed accumulation rates of external metabolites can yield flux distribution at steady state. One such methodology for quantification is the use of elementary modes, which are minimal set of enzymes connecting external metabolites. Here, we have used a linear objective function subject to elementary modes as constraint to determine the fluxes in the metabolic network of Corynebacterium glutamicum. The feasible phenotypic space was evaluated at various combinations of oxygen and ammonia uptake rates.     

Growth of Corynebacterium glutamicum in glucose-limited continuous cultures under high osmotic pressure. Influence of growth rate on the intracellular accumulation of proline,glutamate and trehalose

In order to determine the response of Corynebacterium glutamicum to osmotic stress under different growth conditions, the bacteria were grown in glucose-limited continuous cultures at osmotic pressures of 0.4–2.4 osmol kg^–1 by addition of NaCl to the culture medium. Steady-state continuous cultures were obtained for all investigated osmotic pressures. Increasing the medium osmolality resulted in a higher specific glucose-uptake rate, a lower glucose-to-biomass conversion yield, as well as important changes in the cellular content. A short-term response to the addition of NaCl to a continuous culture was the rapid but transient uptake of Na⁺ ions. At steady state a higher osmotic pressure resulted in a strong increase of the intracellular concentrations of proline, from 5 mg/g to 125 mg/g dry weight, and of trehalose from 20 mg/g to 60 mg/g dry weight. The level of glutamate, which was the dominant intracellular amino acid at low osmotic pressure at 55 mg/g dry weight, was not affected by the addition of NaCl. The influence of the specific growth rate, between 0.1 h^–1 and 0.4 h^–1, on the intracellular metabolite concentration was also determined. The level of proline was found to increase strongly with the growth rate, whereas the trehalose content decreased slightly and the glutamate content did not change. The observed net increase in accumulated metabolites may be related to a requirement of a higher turgor pressure for rapid cell growth.     

A phenomenological model to represent the kinetics of growth by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for lysine production

Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.     

Elucidation of anaplerotic pathways in Corynebacterium glutamicum via 13C-NMR spectroscopy and GC-MS

We have obtained direct evidence indicating the presence of pyruvate-carboxylating activity in Corynebacterium glutamicum, a lysine-overproducing bacterium. This evidence was obtained through the use of ¹³C nuclear magnetic resonance (NMR) spectroscopy and gas chromatography/mass spectrometry (GC-MS) of secreted metabolites in a lysine fermentation. The distribution of ¹³C label after multiple turns in the tricarboxylic acid cycle was accounted for properly to obtain predictions for [¹³C] metabolite enrichments that were employed in the interpretation of ¹³C-NMR and GC-MS data. Of critical importance in arriving at the conclusions was the use of C. glutamicum mutants with deletions of the pyruvate kinase and/or phosphoenolpyruvate carboxylase enzymes. Our results demonstrate the presence of pyruvate-carboxylating pathway(s) in C.␣glutamicum operating simultaneously with phosphoenolpyruvate carboxylase, with the latter enzyme contributing approximately 10 % of the total oxaloacetate synthesis during the lysine-production phase with pyruvate and gluconate as carbon sources. These findings are important for developing strategies to increase the total carbon flux for synthesis of amino acids of the aspartate family through metabolic engineering. Received: 11 June 1996 / Received revision: 30 October 1996 / Accepted: 15 November 1996     

Carbon flux analysis in a pantothenate overproducing Corynebacterium glutamicum strain

Carbon flux analysis during a pseudo-stationary phase of metabolite accumulation in a genetically engineered strain of Corynebacterium glutamicum, containing plasmids leading to over-expression of the ilvBNCD and panBC operons, has identified the basic metabolic constraints governing the potential of this bacterium to produce pantothenate. Carbon flux converging on pyruvate (75% of glucose uptake) is controlled by anabolic precursor requirements and NADPH demand provoking high carbon loss as CO₂ via the pentose pathway. Virtually all the flux of pyruvate is directed into the branched pathway leading to both valine and pantothenate production, but flux towards valine is tenfold higher than that transformed to pantothenate, indicating that significant improvements will only be obtained if carbon flux at the ketoisovalerate branchpoint can be modulated.     

Response to osmotic stress and temperature of the fungus <Emphasis Type="Italic">Ustilago maydis</Emphasis>

Ustilago maydis is a fungal pathogen which is exposed during its life cycle to both abiotic and biotic stresses before and after the infection of maize. To cope with extreme environmental changes, microorganisms usually accumulate the disaccharide trehalose. We have investigated both the accumulation of trehalose and the activity of trehalase during the adaptation of U. maydis haploid cells to thermal, sorbitol, and NaCl stresses. Sorbitol and sodium chloride induced sustained accumulation of trehalose, while a transient increase was observed under heat stress. Sorbitol stressed cells showed higher trehalase activity compared with control cells and to those stressed by NaCl and high temperature. Addition of cycloheximide, a protein synthesis inhibitor, did not affect the trehalose accumulation during the first 15 min, but basal levels of trehalose were reached after the second period of 15 min. The proteomic analysis of the response of U. maydis to temperature, sorbitol, and salt stresses indicated a complex pattern which highlights the change of 18 proteins involved in carbohydrate and amino acid metabolism, protein folding, redox regulation, ion homeostasis, and stress response. We hypothesize that trehalose accumulation during sorbitol stress in U. maydis might be related to the adaptation of this organism during plant infection.     

Osmotic Stress Response: Quantification of Cell Maintenance and Metabolic Fluxes in a Lysine-Overproducing Strain of Corynebacterium glutamicum

Osmotic stress diminishes cell productivity and may cause cell inactivation in industrial fermentations. The quantification of metabolic changes under such conditions is fundamental for understanding and describing microbial behavior during bioprocesses. We quantified the gradual changes that take place when a lysine-overproducing strain of Corynebacterium glutamicum is grown in continuous culture with saline gradients at different dilution rates. The use of compatible solutes depended on environmental conditions; certain osmolites predominated at different dilution rates and extracellular osmolalities. A metabolic flux analysis showed that at high dilution rates C. glutamicum redistributed its metabolic fluxes, favoring energy formation over growth. At low dilution rates, cell metabolism accelerated as the osmolality was steadily increased. Flexibility in the oxaloacetate node proved to be key for the energetic redistribution that occurred when cells were grown at high dilution rates. Substrate and ATP maintenance coefficients increased 30- and 5-fold, respectively, when the osmolality increased, which demonstrates that energy pool management is fundamental for sustaining viability.     

Trehalose metabolism in Escherichia coli: stress protection and stress regulation of gene expression

Endogenously synthesized trehalose is a stress protectant in Escherichia coli. Externally supplied trehalose does not serve as a stress protectant, but it can be utilized as the sole source of carbon and energy. Mutants defective in trehalose synthesis display an impaired osmotic tolerance in minimal growth media without glycine betaine, and an impaired stationary-phaseinduced heat tolerance. Mechanisms for stress protection by trehalose are discussed. The genes for trehalose-6-phosphate synthase (otsA) and anabolic trehalose-6-phosphate phosphatase (otsB) constitute an operon. Their expression is induced both by osmotic stress and by growth into the stationary phase and depend on the sigma factor encoded by rpoS (katF). rpoS is amber-mutated in E. coli K-12 and its DNA sequence varies among K-12 strains. For trehalose catabolism under osmotic stress E. coli depends on the osmoticcally inducible periplasmic trehalase (TreA). In the absence of osmotic stress, trehalose induces the formation of an enzyme II^Tre (TreB) of the group translocation system, a catabolic trehalose-6-phosphate phosphatase (TreE), and an amylotrehalase (TreC) which converts trehalose to free glucose and a glucose polymer.     

Trehalose determination in linseed subjected to osmotic stress. HPAEC‐PAD analysis: an inappropriate method

Trehalose is a non‐reducing disaccharide involved in stress tolerance in plants. To understand better the role of trehalose in the osmotic stress response in linseed (Linum usitatissimum), trehalose content in leaves was studied. First, the method commonly used for sugar determination, high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD), gave unsatisfactory results and the separation efficiency could not be improved by varying the elution conditions. The same problem was also found in the model plant: Arabidopsis thaliana. After clearly highlighting a co‐elution of trehalose in these two species by a trehalase assay and liquid chromatography‐high resolution mass spectrometry analysis, gas chromatography–mass spectrometry (GC‐MS) was used as the analytical method instead. These results confirmed that trehalose content is currently overestimated by HPAEC‐PAD analysis, approximately 7 and 13 times for A. thaliana and linseed respectively. Thus GC‐MS gave more satisfactory results for trehalose quantification in plants. With this method, trehalose accumulation was observed in linseed during an osmotic stress (?0.30 MPa), the quantity (31.49 nmol g^–1 dry weight after 48 h) appears too low to assign an osmoprotector or osmoregulator role to trehalose in stressed linseed.     

Comparative Metabolic Flux Analysis of Lysine-Producing Corynebacterium glutamicum Cultured on Glucose or Fructose

A comprehensive approach to ¹³C 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.