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L Eggeling

Publications and source records attributed to L Eggeling.

At least 19 recordsLinked to original sources
PubMed2002

3-Phosphoglycerate dehydrogenase from Corynebacterium glutamicum: the C-terminal domain is not essential for activity but is required for inhibition by L-serine.

The serA gene of Corynebacterium glutamicum coding for 3-phosphoglycerate dehydrogenase (PGDH) was isolated and functionally characterized. It encodes a polypeptide of 530 aminoacyl residues (aa), which is substantially longer than the corresponding Escherichia coli polypeptide of 410 aa. The difference is largely due to an additional stretch of aa in the carboxy- (C)-terminal part of the polypeptide. Overexpression of serA in C. glutamicum results in a 16-fold increase in specific PGDH activity to 2.1 U/mg protein, with activity being inhibited by high concentrations of L-serine. A set of muteins that were progressively truncated at the C-terminal end was constructed. When overexpressed, mutein SerADelta197 showed a specific PGDH dehydrogenase activity of 1.3 U/mg protein, with the activity no longer being sensitive to L-serine. Gel filtration experiments showed that wild type PGDH is a homotetramer, whereas mutein SerADelta197 constitutes a dimer. Thus, the specific regulatory features of C. glutamicum PGDH are due to the C-terminal part of the polypeptide, which can be deleted with almost no effect on the catalytic activity of the enzyme.

Carbohydrate Dehydrogenases↗
PubMed2002

Influence of threonine exporters on threonine production in Escherichia coli.

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.

Amino Acid Transport Systems, Neutral↗
PubMed2002

Expression of genes of lipid synthesis and altered lipid composition modulates L-glutamate efflux of Corynebacterium glutamicum.

L-Glutamate is made with Corynebacterium glutamicum on a scale of more than 106 tons/year. Nevertheless, formation of this amino acid is enigmatic and there is very limited molecular information available to unravel the apparently complex conditions leading to L-glutamate efflux. Here, we report the isolation and overexpression of the genes involved in lipid synthesis: acp, fadD 15, cma, cls, pgsA2, cdsA, gpsA, and plsC, and the inactivation of cma and cls. In addition, the consequences for phospholipid content, temperature sensitivity, as well as detergent-independent and detergent-dependent L-glutamate efflux were quantified. An in part strong alteration of the phospholipid composition was achieved; for instance, overexpression offadD15 encoding an acyl-CoA ligase resulted in an increase of phosphatidyl inositol from 12.6 to 30.2%. All strains, except that overexpressing acp (acyl carrier protein), exhibited increased temperature sensitivity, with the strongest sensitivity present upon cls (cardiolipin synthetase) inactivation. As a consequence of the genetically modified lipid synthesis, L-glutamate efflux changed quite dramatically; for instance, overexpression of plsC (acylglycerolacyl transferase) resulted in a detergent-triggered increase of L-glutamate accumulation from 92 mM to 108 mM, whereas acp overexpression reduced the accumulation to 24 mM. With some of the overexpressed genes, substantial L-glutamate excretion even without detergent addition was obtained when the fermentation temperature was elevated. These data show that the chemical and physical properties of the cytoplasmic membrane are altered and suggest that this is a necessary precondition to achieve L-glutamate efflux.

Bacterial Proteins↗
PubMed2001

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.

Corynebacterium↗
PubMed2001

Expression control and specificity of the basic amino acid exporter LysE of Corynebacterium glutamicum.

LysE of Corynebacterium glutamicum belongs to a large new superfamily of translocators whose members are probably all involved in the export of small solutes. Here, the transcript initiation site of lysE, and its divergently transcribed regulator gene, lysG, are identified. Single-copy transcriptional fusions of lysE with lacZ, and titration experiments, show that LysG is the positive regulator of lysE expression enabling its up to 20-fold induction. This induction requires the presence of a coinducer, which is either intracellular L-lysine, or L-arginine. A competition experiment showed that LysE exports these two basic amino acids at comparable rates of about 0.75 nmol min(-1) (mg dry wt)(-1). Although L-histidine and L-citrulline also act as coinducers of lysE expression, these two amino acids are not exported by LysE. As is evident from the analysis of a lysEG deletion mutant, the physiological role of the lysEG system is to prevent bacteriostasis due to elevated L-lysine or L-arginine concentrations that arise during growth in the presence of peptides or in mutants possessing a deregulated biosynthesis pathway. C. glutamicum has additional export activities other than those of LysE for exporting L-histidine, L-citrulline and L-ornithine.

Amino Acid Transport Systems, Basic↗
PubMed2001

L-threonine export: use of peptides to identify a new translocator from Corynebacterium glutamicum.

Bacterial mechanisms for the uptake of peptides and their hydrolysis to amino acids are known in great detail, whereas much less is known about the fates of the peptide-derived amino acids. We show that the addition of L-threonine-containing di- or tripeptides results in reduction of the growth of Corynebacterium glutamicum, with concomitant high intracellular accumulation of L-threonine to up to 130 mM. Using transposon mutagenesis and isolation of mutants with increased Thr peptide sensitivity, nine open reading frames (ORFs) were identified, almost all encoding hypothetical proteins of unknown function. Three ORFs encode membrane proteins. Their individual functional characterizations in the wild-type background led to the identification of thrE. Upon thrE overexpression, growth is no longer sensitive to the presence of the Thr peptide, and L-threonine is exported at a rate of 3.8 nmol min(-1) mg of dry weight(-1), whereas the rate of export of a thrE inactivation mutant is reduced to 1.1 nmol min(-1) mg of dry weight(-1). In addition to L-threonine, L-serine is also a substrate for the exporter. The exporter exhibits nine predicted transmembrane-spanning helices with long charged C and N termini and with an amphipathic helix present within the N terminus. All these data suggest that the carrier encoded by thrE serves to export small molecules such as L-threonine and that the carrier is a prototype of a new translocator family. Homologues of ThrE are present in Mycobacterium tuberculosis and Streptomyces coelicolor.

Amino Acid Sequence↗
PubMed2000

In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum.

The C(3)-C(4) metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. C(3) carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the same time multiple C(4)-decarboxylating enzymes may be present. The functions of such parallel reactions are not yet fully understood. Using a (13)C NMR-based strategy, we here quantify the individual fluxes at the anaplerotic node of Corynebacterium glutamicum, which is an example of a bacterium possessing multiple carboxylation and decarboxylation reactions. C. glutamicum was grown with a (13)C-labeled glucose isotopomer mixture as the main carbon source and (13)C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15 positional labels of biomass compounds were quantified. Applying a generally applicable mathematical model to include metabolite mass and carbon labeling balances, it is shown that pyruvate carboxylase contributed 91 +/- 7% to C(3) carboxylation. The total in vivo carboxylation rate of 1.28 +/- 0.14 mmol/g dry weight/h exceeds the demand of carboxylated metabolites for biosyntheses 3-fold. Excess oxaloacetate was recycled to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. This shows that the reactions at the anaplerotic node might serve additional purposes other than only providing C(4) metabolites for biosynthesis.

Amino Acids↗
PubMed2000

Attenuation control of ilvBNC in Corynebacterium glutamicum: evidence of leader peptide formation without the presence of a ribosome binding site.

The ilvBNC operon of Corynebacterium glutamicum encodes acetohydroxy acid synthase and isomero-reductase, which are key enzymes of L-isoleucine, L-valine and L-leucine syntheses. In this study we identified the transcript initiation site of ilvBNC operon 292 nucleotides in front of the first structural gene, and detected the formation of a short transcript from the leader region in addition to the full-length transcript of the operon. This identifies the control of ilvBNC transcription by an attenuation mechanism involving antitermination. Mutations in the leader region were made and their effect on the operon expression in ilvB'lacZ fusions was quantified. Although a presumed leader-peptide-coding region is only one nucleotide away from the transcript initiation site determined, there is clear evidence to support the formation of this leader peptide: (i) the substitution of initiation codon ATG of the peptide by AGG reduced lacZ expression of the appropriate fusion construct to 19%; (ii) the replacement of three subsequent Val codons by Ala codons resulted in the loss of Val-dependent expression; and (iii) a leader peptide LacZ fusion resulted in active beta-galactosidase. Based on these results, it is concluded that transcription of ilvBNC is controlled by a translational-coupled attenuation mechanism. The absence of a ribosome binding site for leader peptide formation means that additional mechanisms may contribute to the transcription control at the decoding initiation step in the leader peptide formation.

Journal Article↗
PubMed2000

Characterization of a bordetella pertussis diaminopimelate (DAP) biosynthesis locus identifies dapC, a novel gene coding for an N-succinyl-L,L-DAP aminotransferase.

The functional complementation of two Escherichia coli strains defective in the succinylase pathway of meso-diaminopimelate (meso-DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs). In line with the successful complementation of the E. coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E. coli and many other bacteria which exhibit tetrahydrodipicolinate succinylase and N-succinyl-L,L-DAP desuccinylase activity, respectively. The first ORF within the operon showed significant sequence similarities with transaminases and contains the characteristic pyridoxal-5'-phosphate binding motif. Enzymatic studies revealed that this ORF encodes a protein with N-succinyl-L,L-DAP aminotransferase activity converting N-succinyl-2-amino-6-ketopimelate, the product of the succinylase DapD, to N-succinyl-L,L-DAP, the substrate of the desuccinylase DapE. Therefore, this gene appears to encode the DapC protein of B. pertussis. Apart from the pyridoxal-5'-phosphate binding motif, the DapC protein does not show further amino acid sequence similarities with the only other known enzyme with N-succinyl-L,L-DAP aminotransferase activity, ArgD of E. coli.

Acyltransferases↗
PubMed1999

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.

Corynebacterium↗
PubMed1999

D-Pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction.

D-Pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. We quantified three of these enzyme activities in Corynebacterium glutamicum and determined specific activities ranging from 0.00014 to 0.001 micromol/min mg (protein)-1. The genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. These studies suggest that panBC constitutes an operon. By using panC, an assay system was developed to quantify D-pantothenate. The wild type of C. glutamicum was found to accumulate 9 micrograms of this vitamin per liter. A strain was constructed (i) to abolish L-isoleucine synthesis, (ii) to result in increased ketoisovalerate formation, and (iii) to enable its further conversion to D-pantothenate. The best resulting strain has ilvA deleted from its chromosome and has two plasmids to overexpress genes of ketoisovalerate (ilvBNCD) and D-pantothenate (panBC) synthesis. With this strain a D-pantothenate accumulation of up to 1 g/liter is achieved, which is a 10(5)-fold increase in concentration compared to that of the original wild-type strain. From the series of strains analyzed it follows that an increased ketoisovalerate availability is mandatory to direct the metabolite flux into the D-pantothenate-specific part of the pathway and that the availability of beta-alanine is essential for D-pantothenate formation.

Base Sequence↗
PubMed1999

The LysE superfamily: topology of the lysine exporter LysE of Corynebacterium glutamicum, a paradyme for a novel superfamily of transmembrane solute translocators.

In Corynebacterium glutamicum the LysE carrier protein exhibits the unique function of exporting L-lysine. We here analyze the membrane topology of LysE, a protein of 236 amino acyl residues, using PhoA- and LacZ-fusions. The amino-terminal end of LysE is located in the cytoplasm whereas the carboxy-terminal end is found in the periplasm. Although 6 hydrophobic domains were identified based on hydropathy analyses, only five transmembrane spanning helices appear to be present. The additional hydrophobic segment may dip into the membrane or be surface localized. We show that LysE is a member of a family of proteins found, for example, in Escherichia coil, Bacillus subtilis, Mycobacterium tuberculosis and Helicobacter pylori. This family, which we have designated the LysE family, is distantly related to two additional protein families which we have designated the YahN and CadD families. These three families, the members of which exhibit similar sizes, hydropathy profiles, and sequence motifs comprise the LysE superfamily. Functionally characterized members of the LysE superfamily export L-lysine, cadmium and possibly quarternary amines. We suggest that LysE superfamily members will prove to catalyze export of a variety of biologically important solutes.

Amino Acid Sequence↗
PubMed1998

Urea uptake and urease activity in Corynebacterium glutamicum.

When Corynebacterium glutamicum is grown with a sufficient nitrogen supply, urea crosses the cytoplasmic membrane by passive diffusion. A permeability coefficient for urea diffusion of 9 x 10(-7) cm s-1 was determined. Under conditions of nitrogen starvation, an energy-dependent urea uptake system was synthesized. Carrier-mediated urea transport was catalyzed by a secondary transport system linked with proton motive force. With a Km for urea of 9 microM, the affinity of this uptake system was much higher than the affinity of urease towards its substrate (Km approximately 55 mM urea). The maximum uptake velocity depended on the expression level and was relatively low [2-3.5 nmol min-1 (mg dry wt.)-1].

Adenosine Triphosphate↗
PubMed1998

Improved L-lysine yield with Corynebacterium glutamicum: use of dapA resulting in increased flux combined with growth limitation.

The amino acid L-lysine is produced on a large scale using mutants of Corynebacterium glutamicum. However, as yet recombinant DNA techniques have not succeed in improving strains selected for decades by classic mutagenesis for high productivity. We here report that seven biosynthetic enzymes were assayed and oversynthesis of the dihydrodipicolinate synthase resulted in an increase of lysine accumulation from 220 mM to 270 mM. The synthase, encoded by dapA, is located at the branch point of metabolite distribution to either lysine or threonine and competes with homoserine dehydrogenase for the common substrate aspartate semialdehyde. When graded dapA expression was used, as well as quantification of enzyme activities, intracellular metabolite concentrations and flux rates, a global response of the carbon metabolism to the synthase activity became apparent: the increased flux towards lysine was accompanied by a decreased flux towards threonine. This resulted in a decreased growth rate, but increased intracellular levels of pyruvate-derived valine and alanine. Therefore, modulating the flux at the branch point results in an intrinsically introduced growth limitation with increased intracellular precursor supply for lysine synthesis. This does not only achieve an increase in lysine yield but this example of an intracellularly introduced growth limitation is proposed as a new general means of increasing flux for industrial metabolite over-production.

Corynebacterium↗
PubMed1998

Analysis of the leuB gene from Corynebacterium glutamicum.

The leuB gene of Corynebacterium glutamicum was found to be present on a 2.2-kb BamHI-SacI chromosomal fragment which complemented the leuB mutation of Escherichia coli. The activity of 3-isopropylmalate dehydrogenase (EC 1.1.1.85), encoded by the leuB gene, was significantly increased in C. glutamicum cells harbouring a plasmid containing the 2.2-kb fragment. The nucleotide sequence of the C. glutamicum leuB coding region (an open reading frame, ORF, of 1020 bp encoding a polypeptide of 340 amino acids with M(r) of 36 144) was determined. The deduced amino acid sequence of the product of this ORF is highly homologous to those of 3-isopropylmalate dehydrogenases from three species of mycobacteria. The transcriptional start site of the leuB gene was localized 35 bp upstream of its translational start; a functional terminator was detected in the 3' flanking region. Northern hybridization analysis showed that the C. glutamicum leuB gene is transcribed as a single monocistronic RNA (approximately 1.2 kb in size). Activity of the leuB promoter was significantly reduced when leucine was present in the growth medium. This suggests the negative regulation of the leuB expression on the transcriptional level in C. glutamicum cells.

3-Isopropylmalate Dehydrogenase↗
PubMed1998

Different modes of diaminopimelate synthesis and their role in cell wall integrity: a study with Corynebacterium glutamicum.

In eubacteria, there are three slightly different pathways for the synthesis of m-diaminopimelate (m-DAP), which is one of the key linking units of peptidoglycan. Surprisingly, for unknown reasons, some bacteria use two of these pathways together. An example is Corynebacterium glutamicum, which uses both the succinylase and dehydrogenase pathways for m-DAP synthesis. In this study, we clone dapD and prove by enzyme experiments that this gene encodes the succinylase (M(r) = 24082), initiating the succinylase pathway of m-DAP synthesis. By using gene-directed mutation, dapD, as well as dapE encoding the desuccinylase, was inactivated, thereby forcing C. glutamicum to use only the dehydrogenase pathway of m-DAP synthesis. The mutants are unable to grow on organic nitrogen sources. When supplied with low ammonium concentrations but excess carbon, their morphology is radically altered and they are less resistant to mechanical stress than the wild type. Since the succinylase has a high affinity toward its substrate and uses glutamate as the nitrogen donor, while the dehydrogenase has a low affinity and incorporates ammonium directly, the m-DAP synthesis is another example of twin activities present in bacteria for access to important metabolites such as the well-known twin activities for the synthesis of glutamate or for the uptake of potassium.

Acyltransferases↗
PubMed1996

Construction of L-lysine-, L-threonine-, and L-isoleucine-overproducing strains of Corynebacterium glutamicum.

The gram-negative bacterium Corynebacterium glutamicum is used for the industrial production of amino acids, for example, of L-glutamate and L-lysine. By cloning and expressing the various genes of the L-lysine pathway in C. glutamicum, we would demonstrate that an increase of the flux of L-aspartate semialdehyde to L-lysine could be obtained in strains with increased dihydrodipicolinate synthase activity. Recently we detected that in C. glutamicum two pathways exist for synthesis of D,L-diaminopimelate and L-lysine. Mutants defective in one pathway are still able to synthesize enough L-lysine for growth, but the L-lysine secretion is reduced to 50 to 70%. Using NMR spectroscopy, we could calculate how much of the L-lysine secreted into the medium is synthesized via either one or the other pathway. Amplification of the feedback inhibition insensitive homoserine dehydrogenase and homoserine kinase in a high L-lysine-overproducing strain enabled channeling of the carbon flow from the intermediate aspartate semialdehyde towards homoserine, resulting in a high accumulation of L-threonine. For a further flux from L-threonine to L-isoleucine, the allosteric control of threonine dehydratase was eliminated.

Biotechnology↗