A. A. de Graaf

Metabolic state of Zymomonas mobilis in glucose-, fructose-, and xylose-fed continuous cultures as analysed by 13C- and 31P-NMR spectroscopy.

Abstract The reasons for the well-known significantly different behaviour of the anaerobic, gram-negative, ethanologenic bacterium Zymomonas mobilis during growth on fructose (i.e. decreased growth and ethanol yields, increased by-product formation) as compared to that on its second natural substrate, glucose, have remained unexplained. A xylose-fermenting recombinant strain of Z. mobilis that was recently constructed in our laboratory also unexpectedly displayed an increased formation of by-products and a strongly reduced growth rate as compared to the parent strain. Therefore, a comprehensive study employing recently developed NMR-based methods for the in vivo analysis of intracellular phosphorylated pool sizes and metabolic fluxes was undertaken to enable a global characterization of the intracellular metabolic state of Z. mobilis during growth on 13C-labelled glucose, fructose and xylose in defined continuous cultures. The 13C-NMR flux analysis indicated that ribose 5-phosphate is synthesized via the nonoxidative pentose phosphate pathway in Z. mobilis, and it identified a metabolic bottleneck in the recombinant xylose-fermenting Z. mobilis strain at the level of heterologous xylulokinase. The 31P-NMR analyses revealed a global alteration of the levels of intracellular phosphorylated metabolites during growth on fructose as compared to that on glucose. The results suggest that this is primarily caused by an elevated concentration of intracellular fructose 6-phosphate.

13C NMR studies of the fluxes in the central metabolism of Corynebacterium glutamicum during growth and overproduction of amino acids in batch cultures

The carbon flux distribution in the central metabolism of Corynebacterium glutamicum was studied in batch cultures using [1-13C]- and [6-13C]glucose as substrate during exponential growth as well as during overproduction of l-lysine and l-glutamate. Using the 13C NMR data in conjunction with stoichiometric metabolite balances, molar fluxes were quantified and normalised to the glucose uptake rate, which was set to 100. The normalised molar flux via the hexose monophosphate pathway was 40 during exponential growth, whereas it was only 17 during l-glutamate production. During l-lysine production, the normalised hexose monophosphate pathway flux was elevated to 47. Thus, the carbon flux via this pathway correlated with the NADPH demand for bacterial growth and l-lysine overproduction. The normalised molar flux in the tricarboxylic acid cycle at the level of 2-oxoglutarate dehydrogenase was 100 during exponential growth and 103 during l-lysine secretion. During l-glutamate formation, the normalised flux through the tricarboxylic acid cycle was reduced to 60. In contrast to earlier NMR studies with C. glutamicum, no significant activity of the glyoxylate pathway could be detected. All experiments indicated a strong in vivo flux from oxaloacetate back to phosphoenolpyruvate and/or pyruvate, which might be due to phosphoenolpyruvate carboxykinase activity in C. glutamicum.

Metabolic engineering for L-lysine production by Corynebacterium glutamicum.

Corynebacterium glutamicum has been used since several decades for the large-scale production of amino acids, esp. L-glutamate and L-lysine. After initial successes of random mutagenesis and screening approaches, further strain improvements now require a much more rational design, i.e. metabolic engineering. Not only recombinant DNA technology but also mathematical modelling of metabolism as well as metabolic flux analysis represent important metabolic engineering tools. This review covers as state-of-the-art examples of these techniques the genetic engineering of the L-lysine biosynthetic pathway resulting in a vectorless strain with significantly increased dihydrodipicolinate synthase activity, and the detailed metabolic flux analysis by 13C isotopomer labelling strategies of the anaplerotic enzyme activities in C. glutamicum resulting in the identification of gluconeogenic phosphoenolpyruvate carboxykinase as a limiting enzyme.

Development and application of a membrane cyclone reactor for in vivo NMR spectroscopy with high microbial cell densities

A new bioreactor system has been developed for in vivo NMR spectroscopy of microorganisms under defined physiological conditions. This cyclone reactor with an integrated NMR flow cell is continuously operated in the magnet of a 400-MHz wide-bore NMR spectrometer system. The residence times of medium and cells are decoupled by a circulation-integrated cross-flow microfiltration module to achieve higher cell densities as compared to continuous fermentations without cell retention (increase in cell density up to a factor of 10 in steady state). Volumetric mass transfer coefficients k(L)a of more than 1.0 s(-1) are possible in the membrane cyclone reactor, ensuring adequate oxygen supply [oxygen transfer rate >15,000 mg O(2) .(L h)(-1)] of high cell densities. With the aid of the membrane cyclone reactor we were able to show, using continuous in vivo (31)P NMR spectroscopy of anaerobic glucose fermentation by Zymomonas mobilis, that the NMR signal intensity was directly proportional to the cell concentration in the reactor. The concentration profiles of intracellular inorganic phosphate, NAD(H), NDP, NTP, UDP-sugar, a cyclic pyrophosphate, two sugar phosphate pools, and extracellular inorganic phosphate were recorded after a shift from one steady state to another. The intracellular cyclic pyrophosphate had not been detected before in in vitro measurements of Zymomonas mobilis extracts due to the high instability of this compound. Using continuous in vivo (13)C NMR spectroscopy of aerobic glucose utilization by Corynebacterium glutamicum at a density of 25 g(cell dry weight) . L(-1), the membrane cyclone reactor served to measure the different dynamics of labeling in the carbon atoms of L-lactate, L-glutamate, succinate, and L-lysine with a time resolution of 10 min after impressing a [1-(13)C]-glucose pulse.

Oxidative phosphorylation in Zymomonas mobilis

The obligately fermentative aerotolerant bacterium Zymomonas mobilis was shown to possess oxidative phosphorylation activity. Increased intracellular ATP levels were observed in aerated starved cell suspension in the presence of ethanol or acetaldehyde. Ethanolconsuming Z. mobilis generated a transmembrane pH gradient. ATP synthesis in starved Z. mobilis cells could be induced by external medium acidification of 3.5–4.0 pH units. Membrane vesicles of Z. mobilis coupled ATP synthesis to NADH oxidation. ATP synthesis was sensitive to the protonophoric uncoupler CCCP both in starved cells and in membrane vesicles. The H+-ATPase inhibitor DCCD was shown to inhibit the NADH-coupled ATP synthesis in membrane vesicles. The physiological role of oxidative phosphorylation in this obligately fermentative bacterium is discussed.

Production process monitoring by serial mapping of microbial carbon flux distributions using a novel Sensor Reactor approach: II—13C-labeling-based metabolic flux analysis and l-lysine production

Corynebacterium glutamicum is intensively used for the industrial large-scale (fed-) batch production of amino acids, especially glutamate and lysine. However, metabolic flux analyses based on 13C-labeling experiments of this organism have hitherto been restricted to small-scale batch conditions and carbon-limited chemostat cultures, and are therefore of questionable relevance for industrial fermentations. To lever flux analysis to the industrial level, a novel Sensor Reactor approach was developed (El Massaoudi et al., Metab. Eng., submitted), in which a 300-L production reactor and a 1-L Sensor Reactor are run in parallel master/slave modus, thus enabling 13C-based metabolic flux analysis to generate a series of flux maps that document large-scale fermentation courses in detail. We describe the successful combination of this technology with nuclear magnetic resonance (NMR) analysis, metabolite balancing methods and a mathematical description of 13C-isotope labelings resulting in a powerful tool for quantitative pathway analysis during a batch fermentation. As a first application, 13C-based metabolic flux analysis was performed on exponentially growing, lysine-producing C. glutamicum MH20-22B during three phases of a pilot-scale batch fermentation. By studying the growth, (co-) substrate consumption and (by-) product formation, the similarity of the fermentations in production and Sensor Reactor was verified. Applying a generally applicable mathematical model, which included metabolite and carbon labeling balances for the analysis of proteinogenic amino acid 13C-isotopomer labeling data, the in vivo metabolic flux distribution was investigated during subsequent phases of exponential growth. It was shown for the first time that the in vivo reverse C(4)-decarboxylation flux at the anaplerotic node in C. glutamicum significantly decreased (70%) in parallel with threefold increased lysine formation during the investigated subsequent phases of exponential growth.

Monitoring of inorganic polyphosphate dynamics in Corynebacterium glutamicum using a novel oxygen sparger for real time 31P in vivo NMR

For the first time in intact bacterial cells, the dynamics of the build-up of soluble cytosolic inorganic polyphosphate (polyP) during aeration, and its breakdown during anaerobiosis have been observed with a time resolution of 50 s. Under conditions of 60-80% saturation with pure oxygen, the accumulation of high levels of intracellular polyP was detected when inorganic phosphate (Pi) and glucose or acetate were added to Corynebacterium glutamicum cell suspensions (3 ml, ∼40 mg dw/ml). The maximum levels of polyP reached were estimated to 600 mM P units in the cytosol or ∼3% phosphorus [w/w] in the cell dry weight. C. glutamicum polyP was apparently of high molecular weight (containing probably a few hundred units) as inferred from signal distribution, but a temporary average polyP chain length of about n = 40 could be estimated at the initial stages of polyP formation. After each addition of glucose or acetate, oxygen levels followed a steep decline to ∼20% and then an increase to the previous level. In contrast, polyP levels rose after the addition of substrate, and declined again, while the oxygen level recovered. When the oxygen supply was completely switched off, the polyP signal declined immediately, with concomitant re-appearance of phosphomonoester signals (sugar phosphates and related compounds). Both processes, the increase of polyP during aeration and supply with substrate and Pi, and the decrease during anaerobiosis, occurred within minutes. Only within these relatively brief windows of time between successive feedings with substrate or between aeration and anaerobiosis, high levels of polyP could be observed. Thus, our findings indicate that polyP occurs not only as the long known granular storage material in some Corynebacteria, such as C. diphtheriae or C. imitans, but that formation and breakdown of soluble polyP in C. glutamicum is a very dynamic process that may play a decisive role in C. glutamicum and in other strains of this genus. These investigations were made possible by combining nuclear magnetic resonance (NMR) techniques with novel methods of oxygen sparging and online substrate distribution. The sparger was custom made from titanium to fit into 10 mm o.d. NMR tubes. Both the size and the spacing of the holes in the sparger were calculated for optimum distribution of oxygen at 30 °C through 3 ml of C. glutamicum cell suspensions. The experiments were carried out using in vivo31P NMR, and monitoring of oxygen was performed with a miniature oxygen optode in real time. Glucose or acetate and/or phosphate stock solutions could be added in situ.31P NMR analyses of intracellular phosphorus metabolites were sampled with a time resolution of 50 s. The sparger unit, including optode and supply lines, could be easily switched from one sample to another after completion of an experiment. It is suggested to use these analytical tools to investigate other bacterial strains and even cell extracts, shedding further light on the novel roles of polyP in living cells [Schroder, H. C., Muller, W. E. G., (eds.). Inorganic polyphosphates - biochemistry, biology, biotechnology. Prog. Mol. Subcell. Biol. 23 (1999). Springer-Verlag, Berlin].

Using 13C‐nmr spectroscopy to evaluate the binding mechanism of bound pesticide residues in soils: I. Solution high resolution nmr spectroscopy

Soil-bound residues of organochemicals and their metabolites can be extracted together with the humic acid fraction. These residues are only detectable by using radioactive labelling, e.g. 14 C (or 3 H). An analysis of the character of the bonding can be achieved by means of 13 C-NMR spectroscopy. A prerequisite is a 13 C-enrichment of the observed molecule and, if possible, a 13 C-depletion of the humic substances. The fungicide anilazine is known to form high amounts of soil-bound residues within a few days. 13 C-NMR spectra of extracted humic acids of two different orthic luvisols (Parabraunerde), a gleyic cambisol (Pseudogley Braunerde) and of an artificially prepared soil from humified, 13 C-depleted maize straw show ester or ether bonds of anilazine to the humic acids. Line broadening of ≤ 20 Hz yields a group of signals which indicate that different functional groups of the humic acids are responsible bondings

Using 13C‐NMR spectroscopy to evaluate the binding mechanism of bound pesticide residues in soils

Abstract Soil‐bound residues of organochemicals and their metabolites can be extracted together with the humic acid fraction. These residues are only detectable by using radioactive labelling, e.g. 14C (or 3H). An analysis of the character of the bonding can be achieved by means of 13C‐NMR spectroscopy. A prerequisite is a 13C‐enrichment of the observed molecule and, if possible, a 13C‐depletion of the humic substances. The fungicide anilazine is known to form high amounts of soil‐bound residues within a few days. 13C‐NMR spectra of extracted humic acids of two different orthic luvisols (Parabraunerde), a gleyic cambisol (Pseudogley Braunerde) and of an artificially prepared soil from humified, 13C‐depleted maize straw show ester or ether bonds of anilazine to the humic acids. Line broadening of ≤ 20 Hz yields a group of signals which indicate that different functional groups of the humic acids are responsible for the bondings.

Interactive Evaluation of NMR Spectra From in Vivo Isotope Labelling Experiments

Abstract In vivo isotope experiments are of great importance for the quantification of metabolic fluxes. The fractional labelling of intracellular metabolites, that has to be measured for flux estimation, must be computed from an in vivo NMR spectrum by determination of peak areas. This task is extremely difficult because peak parameters are unknown under non standard measurement conditions. Moreover overlapping peaks occur frequently and low signal to noise ratios are encountered. A complementary set of spectral analysis tools is presented, that can solve the spectral decomposition problem. The program DOSIS integrates these tools into an interactive software framework.