Unbalance of L-lysine flux in Corynebacterium glutamicum and its use for the isolation of excretion-defective mutants.

We found that the simple addition of L-methionine to the wild type of Corynebacterium glutamicum results in excretion of the cellular building block L-lysine up to rates of 2.5 nmol/min/mg (dry weight). Biochemical analyses revealed that L-methionine represses the homoserine dehydrogenase activity and reduces the intracellular L-threonine level from 7 to less than 2 mM. Since L-lysine synthesis is regulated mainly by L-threonine (plus L-lysine) availability, the result is enhanced flux towards L-lysine. This indicates a delicate and not well controlled type of flux control at the branch point of aspartate semialdehyde conversion to either L-lysine or L-threonine, probably due to the absence of isoenzymes in C. glutamicum. The inducible system of L-lysine excretion discovered was used to isolate mutants defective in the excretion of this amino acid. One such mutant characterized in detail accumulated 174 mM L-lysine in its cytosol without extracellular excretion of L-lysine, whereas the wild type accumulated 53 mM L-lysine in the cytosol and 5.9 mM L-lysine in the medium. The mutant was unaffected in L-lysine uptake or L-isoleucine or L-glutamate excretion, and also the membrane potential was unaltered. This mutant therefore represents a strain with a defect in an excretion system for the primary metabolite L-lysine.     

Mechanism and Regulation of Isoleucine Excretion in Corynebacterium glutamicum

Whole cells of Corynebacterium glutamicum were loaded with high cytoplasmic l-isoleucine concentrations, and isoleucine excretion from these cells was studied in terms of mechanism and regulation. The transmembrane isoleucine flux could be differentiated into carrier-mediated uptake, carrier-mediated excretion, and diffusion. After discrimination from the other transmembrane solute movements, the outward-directed flux, which was due to the activity of the isoleucine excretion carrier, was characterized with respect to its energy dependence and its regulation at the level of expression. Isoleucine excretion was shown to function as a secondary transport process, driven by the membrane potential and coupled to the movement of protons, presumably with a stoichiometry of 2:1 (H(sup+)/isoleucine). Of a variety of putative transport substrates, only leucine was able to compete for isoleucine at the cis (cytosolic) side of the export carrier. Cytoplasmic isoleucine concentrations higher than 20 mM induce the activity of the isoleucine excretion system. This effect is specific for isoleucine and is inhibited by the presence of chloramphenicol. Apart from leucine, other amino acids and related amino acid analogs are not able to induce isoleucine excretion. The complex pattern of regulation of the isoleucine excretion system at the level of activity and expression is shown to be related to the pattern of regulation of the isoleucine uptake system in C. glutamicum in terms of physiological significance.     

Corynebacterium glutamicum as a host for synthesis and export of D-Amino Acids

A number of d-amino acids occur in nature, and there is growing interest in their function and metabolism, as well as in their production and use. Here we use the well-established l-amino-acid-producing bacterium Corynebacterium glutamicum to study whether d-amino acid synthesis is possible and whether mechanisms for the export of these amino acids exist. In contrast to Escherichia coli, C. glutamicum tolerates d-amino acids added extracellularly. Expression of argR (encoding the broad-substrate-specific racemase of Pseudomonas taetrolens) with its signal sequence deleted results in cytosolic localization of ArgR in C. glutamicum. The isolated enzyme has the highest activity with lysine (100%) but also exhibits activity with serine (2%). Upon overexpression of argR in an l-arginine, l-ornithine, or l-lysine producer, equimolar mixtures of the d- and l-enantiomers accumulated extracellularly. Unexpectedly, argR overexpression in an l-serine producer resulted in extracellular accumulation of a surplus of d-serine (81 mM d-serine and 37 mM l-serine) at intracellular concentrations of 125 mM d-serine plus 125 mM l-serine. This points to a nonlimiting ArgR activity for intracellular serine racemization and to the existence of a specific export carrier for d-serine. Export of d-lysine relies fully on the presence of lysE, encoding the exporter for l-lysine, which is apparently promiscuous with respect to the chirality of lysine. These data show that d-amino acids can also be produced with C. glutamicum and that in special cases, due to specific carriers, even a preferential extracellular accumulation of this enantiomer is possible.     

Occurrence of a novel yeast enzyme, L-lysine epsilon-dehydrogenase, which catalyses the first step of lysine catabolism in Candida albicans

The yeast Candida albicans is able to utilize L-lysine as the sole nitrogen and carbon source accompanied by intracellular accumulation of alpha-aminoadipate-delta-semialdehyde. A novel yeast amino acid dehydrogenase catalysing the oxidative deamination of the epsilon-group of L-lysine was found in this yeast. The enzyme, L-lysine epsilon-dehydrogenase, is strongly induced in cells grown on L-lysine as the sole nitrogen source. The enzyme is specific for both L-lysine and NADP+. The Km values were determined to be 0.87 mM for L-lysine and 0.071 mM for NADP+. An apparent Mr of 87,000 was estimated by gel filtration. The enzyme has maximum activity at pH 9.5 and a temperature optimum of 32 degrees C under our assay conditions.     

Lysine excretion by Corynebacterium glutamicum. 2. Energetics and mechanism of the transport system.

Lysine excretion in Corynebacterium glutamicum was characterized as secondary transport process. It is modulated by three forces: the membrane potential, the chemical potential of lysine, and the proton gradient. The ATP content of the cells did not correlate with the export activity. Lysine is excreted in symport with presumably two OH- ions which is not distinguishable experimentally from an antiport mechanism against two protons. The substrate-loaded carrier is uncharged. When the external substrate concentration is low and no proton gradient present, reorientation of the positively charged, unloaded carrier is rate-limiting. Export then depends on the membrane potential. When the external substrate is high, translocation of the loaded, uncharged carrier is rate-limiting, and export is not modulated by the membrane potential. The lysine secretion system in C. glutamicum is shown to be well adapted to the requirements of metabolite export.     

Metabolic characterization of a L-lysine-producing strain by continuous culture

Continuous culture experiments with the L-producer, Corynebacterium glutamicum, were carried out to characterize the effect of specific growth rate on fermentation yields, specific rates, productivities, and fluxes through the primary metabolism. The specific productivity of L-lysine exhibited a maximum with respect to specific growth rate, with an initial growth-associated behavior up to specific growth rates of about 0.1 h(-1), and a constant specific productivity for specific growth rates in the range of about 0.1 to 0.2 h(-1). The productivity dropped at specific growth rates larger than about 0.2 h(-1). The yield of L-lysine on glucose increased approximately linearly with decreasing specific growth rate over the entire range studied, as did the respiratory quotient. A direct relationship was established between the culture respiratory quotient and the L-lysine yield. By explicitly accounting for glucose used for biomass synthesis, it was shown that the strain synthesizes L-lysine with an intrinsic yield, or efficiency, of about 0.41 mol L-lysine/mol glucose, compared with the theoretical yield of 0.75 mol/mol. Metabolic flux modeling based on the continuous culture data suggests that the production of ATP is not likely to be a limiting factor in L-lysine production, and that a high TCA cycle activity, coupled with a tightly controlled split of metabolite flow at the PEP node, is likely the cause of the large discrepancy between theoretical and actual yields in L-lysine fermentations.     

Lysine excretion by Corynebacterium glutamicum. 1. Identification of a specific secretion carrier system

Corynebacterium glutamicum effectively excretes lysine when the internal lysine concentration is elevated. Lysine efflux was investigated using selected mutants which are not able to regulate lysine biosynthesis by feedback inhibition. Secretion of lysine is not the consequence of unspecific permeability of the plasma membrane but is mediated by a secretion carrier which is specific for lysine. Lysine export is characterized by high activation energy and follows Michaelis-Menten type kinetics with an internal Km of 20 mM and a Vmax of 12 nmol.min-1.mg dry cells-1. Excretion can proceed against a preexisting chemical gradient and against the electrical potential, which rules out a previously suggested pore model. Lysine excretion can also be observed in the wild-type strain especially under conditions of peptide uptake. Its possible physiological function may be related to regulation of internal amino acid concentrations under special growth conditions.     

Optimization of sulphide production in an anaerobic continuous biofilm process with sulphate reducing bacteria

Different biofilm reactors for sulphide production by sulphate reducing bacteria were compared in a packed bed reactor and in two suspended carrier biofilm reactors. Lactate was used as carbon source in the experiments. The process was reversibly inhibited by free sulphide at 14 mM. The packed bed reactor was more efficient and less sensitive to changes. The maximum load in the system was 5.3 g sulphate/l.d.     

Quantitative discrimination of carrier-mediated excretion of isoleucine from uptake and diffusion in Corynebacterium glutamicum.

The efflux of isoleucine in whole cells of Corynebacterium glutamicum was studied. The different amino acid fluxes across the plasma membrane were functionally discriminated into passive diffusion, carrier-mediated excretion, and carrier-mediated uptake. Detailed kinetic analysis was made possible by controlled variation of internal isoleucine from low concentrations to 100 mM by feeding with mixtures of isoleucine-containing peptides. Isoleucine diffusion was experimentally separated and proceeded with a first-order rate constant of 0.083 min-1 or 0.13 microliters.min-1.mg (dry mass)-1, which corresponds to a permeability of 2 x 10(-8) cm.s-1. Uptake of isoleucine was constant at a rate of 1.1 nmol.min-1.mg (dry mass)-1. Carrier-mediated isoleucine excretion was zero below a threshold of 8 mM cytosolic isoleucine. Above this level, a Michaelis-Menten-type kinetics was observed, with a Km of 21 mM (13 mM plus 8 mM threshold value) and a Vmax of 14.5 nmol.min-1.mg (dry mass)-1. The activity of the isoleucine excretion carrier depended on the presence of a membrane potential. Excretion was specific for L-isoleucine (and presumably L-leucine) and could be inhibited by SH reagents.     

Impact of osmotic stress on volume regulation,cytoplasmic solute composition and lysine production in Corynebacterium glutamicum MH20-22B

The response of the L-lysine producing Corynebacterium glutamicum strain MH20-22B to osmotic stress was studied in batch cultures. To mimic the conditions during a fermentation process the long term adaptation of cells subjected to a constant osmotic stress between 1.0 and 2.5 osM was investigated. Cytoplasmic water content and volume of C. glutamicum cells were found to depend on growth phase, extent of osmotic stress and availability of betaine. The maximal cytoplasmic volumes, which were highest at maximal growth rate, were linearily related to osmotic stress, whereas in stationary cells no active volume regulation was observed. Under severe osmotic stress proline was the prominent compatible solute in growing cells. Uptake of betaine, if available in the medium, reduced the concentration of proline from 750 to 300 mM, indicating that uptake of compatible solutes is preferred to synthesis. Furthermore, betaine was shown to have a higher efficiency to counteract osmotic stress, since the overall concentration of compatible solutes was lower in the presence of betaine. Under severe osmotic stress, the addition of betaine shifted L-lysine production in MH20-22B to earlier fermentation times and increased both product concentration and yield in these phases, but did not improve the final L-lysine yield.     

Determining specific biomass activity in anaerobic wastewater treatment processes

An experimental method for the measurement of specific gas production rate was developed and tested with biomass samples taken from anaerobic fluidized bed reactors, operating with a variety of carriers with molasses, condensate from cellulose production and brewery wastewater as feeds. The method is based on reactor sampling and offline gas volume measurement during a known time interval. Important factors are biomass and liquid sampling under oxygen-free conditions, using the liquid from the reactor as substrate, providing sufficient mixing and maintaining the physical integrity of the biomass. The method was developed in such a way that small samples (20 ml) were taken under anaerobic conditions (poising agent) for short-term (2–3 min.) gas rate measurements in a small fluidized bed (25 ml) batch reactor with U-tube. Biomass content was measured by an instrumental nitrogen method (Dumas), followed by weight determination of the carrier. The gas rates measured with the test system, and their dependence on substrate concentration, were in good agreement with those directly measured from the continuous fluidized bed reactor. Additions of molasses and acetate to the sample proved that the influence of concentration on the biomass activity can be obtained only by operating the continuous reactor at the concentration levels of interest. Comparison between the reactors showed large differences in the specific activity and the total reactor activity. It was found when comparing two reactors, that the values of the specific and the total activities permitted the calculation of the relative biomass quantities. In this way the influence of the carrier-type could be evaluated.     

Advanced start-up of anaerobic attached film expanded bed reactor by pre-aeration of biofilm carrier

The start-up and performance of the anaerobic attached film expanded bed (AAFEB) reactor with pre-aeration of carrier were investigated. The carriers of the reactors had been aerated for 10 days before they were put into the AAFEB reactors. The results indicated that the reactors advance the start-up by 15 days, and maintain higher efficiency when they were subjected to organic and hydraulic loading shock, but during steady-state operation, the reactors did not show better performance than the control reactors without pre-aeration of carrier. The thicker biofilm and higher biomass concentration of the reactors with pre-aeration were observed during the start-up period, but the difference between two types of reactors tapered with the time course, and at the steady-state operation, the difference between two types of reactors on these two parameters was not obvious. Maximum specific methane or acids production rates, dehydrogenase activity and coenzyme F(420) content were continuously higher than those of the control reactors. After running 30 days, filamentous bacteria dominated in the reactors with pre-aeration, whereas the cocci were predominant species in the control reactors. It was suggested that the action of the biofilm is strongly dependent on the biofilm thickness or the biomass concentration in normal circumstances, but under adverse circumstances, such as organic or hydraulic loading shock, the characteristics and activity of the anaerobic granular sludge play key roles on the reactor performance. These results clearly indicated that pre-aeration of carrier favor to enhance the start-up and performance of AAFEB reactor.     

Glutamate excretion as a major kinetic bottleneck for the thermally triggered production of glutamic acid by Corynebacterium glutamicum

The study was aimed at evaluating the extent of flux control exercised by the amino acid excretion step on the glutamate production flux in C. glutamicum 2262 strain that is induced for glutamate excretion by an upward temperature shift. Cells initially induced to excrete glutamate were cultivated at different controlled temperatures between 33 and 40 degrees C, and changes in glutamate excretion flux and intracellular concentration were determined in response to increased culture temperature. The fastest growth rate of 0.45 h(-1) and the lowest glutamate excretion rate of 1 mmole/g dw x h were observed at 33 degrees C, together with a high intracellular 0.5 mmole/g dw glutamate accumulation. On the contrary, the fastest glutamate excretion rate of 6 mmole/g dw x h was obtained at 40 degrees C, when cell growth was arrested and the internal glutamate level reduced to 0.25 mmol/g dw. The observed sixfold increase in excretion flux as a result of the temperature increase clearly suggests a specific effect of temperature on the glutamate export system which appears as the major kinetic bottleneck for the glutamate production flux. This conclusion is corroborated by the high internal accumulation of glutamate which, even under the fastest excretion conditions, severely inhibits the activity of the glutamate biosynthesis pathway.     

Effect of transport proteins on l-isoleucine production with the l-isoleucine-producing strain Corynebacterium glutamicum YILW

Previous studies have shown that the deletion of brnQ from the Corynebacterium glutamicum chromosome results in a significant reduction in L-isoleucine uptake rates, while overexpression of brnFE leads to enhanced L-isoleucine export rates. Given that net excretion rates would be an important factor for high titers of L-isoleucine accumulation, we have tested the notion that decreased L-isoleucine uptake combined with increased L-isoleucine excretion will further improve high-yield strains that are currently used for the industrial-scale production of L-isoleucine. To examine the effect of the two carriers on L-isoleucine accumulation in L-isoleucine producer C. glutamicum YILW, we constructed a brnQ deletion mutant (C. glutamicum YILW?brnQ) and two brnFE overexpressors (C. glutamicum YILWpXMJ19brnFE and C. glutamicum YILW?brnQpXMJ19brnFE). Compared to the original strain, the efflux rate of the brnQ mutant increased from 19.0 to 23.6?nmol?min(-1) mg (dry wt)(-1) and its L-isoleucine titer increased from 154.3?mM (20.2?g?l(-1)) to 170.3?mM (22.3?g?l(-1)). The efflux rates of C. glutamicum YILWpXMJ19brnFE and C. glutamicum YILW?brnQpXMJ19brnFE were 33.5 and 39.1?nmol?min(-1) mg (dry wt)(-1), and their L-isoleucine production titers were 197.2?mM (25.9?g?l(-1)) and 221.0?mM (29.0?g?l(-1)), respectively. Our results suggest that modifications of the transport system could provide a promising avenue for further increasing L-isoleucine yield in the L-isoleucine producer.     

Biology of L-lysine overproduction byCorynebacterium glutamicum

Summary The Gram positive bacteriumCorynebacterium glutamicum is used for the production of L-lysine. This review focuses on the progress achieved in the past five years for a deeper understanding of lysine overproduction. This period also coincides with decisive progress in the use of genetic engineering techniques for analysing and increasing the metabolite flux inC. glutamicum. It was thus demonstrated that thein vivo activity of the allosterically controlled aspartate kinase is important for flux control, but in addition also the amount of the dihydrodipicolinate synthase. An outstanding feature ofC. glutamicum is the split lysine biosynthesis pathway. NMR investigations have clearly shown that both pathways are simultaneously usedin vivo and that the flux ratio depends on nitrogen availability. The cellular synthesized lysine is eventually exported into the external medium through a specific carrier. Interestingly lysine producers have other export characteristics so that the carrier properties also seem to be important for increased metabolite flux.     

Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems.

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase. Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.     

Methanogenesis in thermophilic biogas reactors

Methanogenesis in thermophilic biogas reactors fed with different wastes is examined. The specific methanogenic activity with acetate or hydrogen as substrate reflected the organic loading of the specific reactor examined. Increasing the loading of thermophilic reactors stabilized the process as indicated by a lower concentration of volatile fatty acids in the effluent from the reactors. The specific methanogenic activity in a thermophilic pilot-plant biogas reactor fed with a mixture of cow and pig manure reflected the stability of the reactor. The numbers of methanogens counted by the most probable number (MPN) technique with acetate or hydrogen as substrate were further found to vary depending on the loading rate and the stability of the reactor. The numbers of methanogens counted with antibody probes in one of the reactor samples was 10 times lower for the hydrogen-utilizing methanogens compared to the counts using the MPN technique, indicating that other non-reacting methanogens were present. Methanogens that reacted with the probe againstMethanobacterium thermoautotrophicum were the most numerous in this reactor. For the acetate-utilizing methanogens, the numbers counted with the antibody probes were more than a factor of 10 higher than the numbers found by MPN. The majority of acetate utilizing methanogens in the reactor wereMethanosarcina spp. single cells, which is a difficult form of the organism to cultivatein vitro. No reactions were observed with antibody probes raised againstMethanothrix soehngenii orMethanothrix CALS-1 in any of the thermophilic biogas reactors examined. Studies using 2-¹⁴C-labeled acetate showed that at high concentrations (more than approx. 1 mM) acetate was metabolized via the aceticlastic pathway, transforming the methyl-group of acetate into methane. When the concentration of acetate was less than approx. 1 mM, most of the acetate was oxidized via a two-step mechanism (syntrophic acetate oxidation) involving one organism oxidizing acetate into hydrogen and carbon dioxide and a hydrogen-utilizing methanogen forming the products of the first microorganism into methane. In thermophilic biogas reactors, acetate oxidizing cultures occupied the niche ofMethanothrix species, aceticlastic methanogens which dominate at low acetate concentrations in mesophilic systems. Normally, thermophilic biogas reactors are operated at temperatures from 52 to 56° C. Experiments using biogas reactors fed with cow manure showed that the same biogas yield found at 55° C could be obtained at 61° C after a long adaptation period. However, propionate degradation was inhibited by increasing the temperature.     

Startup of anaerobic downflow stationary fixed film (DSFF) reactors

One of the serious problems limiting the application of full-scale anaerobic fixed film processes is reactor startup. To better understand startup, studies with downflow stationary fixed film (DSFF) reactors were conducted to characterize the effects of influent concentration, support material, and surface-to-volume ratio on biofilm development and overall reactor performance. Materials with roughened surfaces gave the best startup performance and as expected increased surface area in the reactors led to more rapid increases in loading rates and higher ultimate loadings. Soluble influent COD concentrations between 5 x 10(3) and 2 x 10(4) mg/L influenced the rate of biofilm development. Lower COD concentrations resulted in faster development of the biofilm, even though ultimate loadings were not necessarily achieved as rapidly as in reactors fed higher strength wastes. No decrease in specific activity of the biofilms in each reactor was observed as the thickness of the biofilms increased to their maximum value at the ultimate loadings. The operation of reactors fed lower strength wastes was more stable than reactors receiving higher strength feeds at comparable loadings. Biofilm yield and activity, COD removals, suspended growth and activity, and other system parameters are discussed.     

Acidification of methanol-fed anaerobic granular sludge bioreactors by cobalt deprivation: Induction and microbial community dynamics

The acidification of mesophilic (30 degrees C) methanol-fed upflow anaerobic sludge bed (UASB) reactors induced by cobalt deprivation from the influent was investigated by coupling the reactor performance (pH 7.0; organic loading rate 4.5 g COD . L(-1) . d(-1)) to the microbial ecology of the bioreactor sludge. The latter was investigated by specific methanogenic activity (SMA) measurements and fluorescence in situ hybridization (FISH) to quantify the abundance of key organisms over time. This study hypothesized that under cobalt limiting conditions, the SMA on methanol of the sludge gradually decreases, which ultimately results in methanol accumulation in the reactor effluent. Once the methanol accumulation surpasses a threshold value (about 8.5 mM for the sludge investigated), reactor acidification occurs because acetogens outcompete methylothrophic methanogens at these elevated methanol concentrations. Methanogens present in granular sludge at the time of the acidification do not use methanol as the direct substrate and are unable to degrade acetate. Methylotrophic/acetoclastic methanogenic activity was found to be lost within 10 days of reactor operation, coinciding with the disappearance of the Methanosarcina population. The loss of SMA on methanol can thus be used as an accurate parameter to predict reactor acidification of methanol-fed UASB reactors operating under cobalt limiting conditions.