The efficient production of L-glutamate is dependent on the product's rapid efflux, hence researchers have recently concentrated on artificially modifying its transport system and cell membrane wall structure. Considering the unique composition and structure of the cell wall of Corynebacterium glutamicum, we investigated the effects of CmpLs on L-glutamate synthesis and transport in SCgGC7, a constitutive L-glutamate efflux strain. First, the knockout strains of CmpLs were constructed, and it was confirmed that the deletion of CmpL1 and CmpL4 significantly improved the performance of L-glutamate producers. Next, temperature-sensitive L-glutamate fermentation with the CmpL1 and CmpL4 knockout strains were carried out in 5 L bioreactors, where the knockout strains showcased temperature-sensitive characteristics and enhanced capacities for L-glutamate production under high temperatures. Notably, the CmpL1 knockout strain outperformed the control strain in terms of L-glutamate production, showing production and yield increases of 69.2% and 55.3%, respectively. Finally, the intracellular and extracellular metabolites collected at the end of the fermentation process were analyzed. The modification of CmpLs greatly improved the L-glutamate excretion and metabolic flux for both L-glutamate production and transport. Additionally, the CmpL1 knockout strain showed decreased accumulation of downstream metabolites of L-glutamate and intermediate metabolites of tricarboxylic acid (TCA) cycle, which were consistent with its high L-glutamate biosynthesis capacity. In addition to offering an ideal target for improving the stability and performance of the industrial strains for L-glutamate production, the functional complementarity and redundancy of CmpLs provide a novel target and method for improving the transport of other metabolites by modification of the cell membrane and cell wall structures in C. glutamicum.
Corynebacterium glutamicum/genetics* ; Glutamic Acid/biosynthesis* ; Fermentation ; Metabolic Engineering ; Bacterial Proteins/metabolism* ; Bioreactors/microbiology* ; Gene Knockout Techniques

In order to enhance gamma-aminobutyric acid production from L-glutamate efficiently, we amplified the key enzyme glutamate decarboxylase (GAD) encoding gene lpgad from the strain Lactobacillus plantarum GB 01-21 which was obtained by way of multi-mutagenesis and overexpressed it in E. coli BL21. Then we purified GAD by Ni-NTA affinity chromatography and characterized the enzyme to optimize the conditions of the whole-cell transformation. The results showed that the recombinant E. coli BL21 (pET-28a-lpgad) produced 8.53 U/mg GAD, which was increased by 3.24 fold compared with the GAD activity in L. plantarum. The optimum pH and temperature of the enzyme were pH 4.8 and 37 degrees C, respectively. At the same time, we found that Ca2+ and Mg2+ could increase the activity significantly. Based on this, we investigated gamma-aminobutyric acid transformation in 5 L fermentor under the optimum transformation conditions. Accordingly, the yield of gamma-aminobutyric acid was 204.5 g/L at 24 h when the 600 g L-glutamate was added and the mole conversion rate had reached 97.92%. The production of gamma-aminobutyric acid was improved by 42.5% compared with that under the unoptimized transformation conditions. This paved a way for the gamma-aminobutyric acid construction of the industrial applications.
Cloning, Molecular ; Escherichia coli ; enzymology ; genetics ; metabolism ; Glutamate Decarboxylase ; biosynthesis ; genetics ; Glutamic Acid ; metabolism ; Lactobacillus plantarum ; enzymology ; genetics ; Recombination, Genetic ; gamma-Aminobutyric Acid ; biosynthesis

A novel method based on pH value was proposed to simplify the substrate feeding method for glutamic acid fermentation. The linear relationship between the consumption amounts of ammonia (x) and that of glucose (y) was established (y = 7.4744x, R2 = 0.9989) which could be used as the ratio of the amount of ammonia and that of glucose in the feeding broth. Thus the concentration of glucose could be controlled through the adjustment of pH automatically. In the glutamic acid fermentation using the pH feedback-controlled glucose feeding method, the glucose concentration in fermentation broth was maintained between 12 and 21 g/L. Compare with the constant glucose concentration feeding method, the glucose conversion rate and glutamic acid productivity increased by 9.06% and 17.5% respectively, when the pH feedback-controlled glucose feeding method was employed, and fermentation period was shorten above 2 h.
Bioreactors ; microbiology ; Corynebacterium glutamicum ; growth & development ; metabolism ; Culture Techniques ; Feedback ; Fermentation ; Glucose ; metabolism ; Glutamic Acid ; biosynthesis ; metabolism ; Hydrogen-Ion Concentration ; Quaternary Ammonium Compounds ; metabolism

OBJECTIVETo investigate the effects of saikosaponin a (SSa) on Glu-activated hippocampal astrocytes of rats.

METHODSNeonatal rat (1-3 days) hippocampal astrocytes were obtained and divided into control group, L-Glu activation group and SSa groups with SSa treatment at 5, 2.5, and 1.25 mg/L. The cell proliferation, cell cycle changes, and expression of glial fibrillary acidic protein (GFAP) after the treatments were assessed with MTT assay, flow cytometry and Western blotting, respectively.

RESULTSIn comparison with Glu-activation group, SSa treatment resulted in significant inhibition of the cell proliferation, cell division and GFAP expression in the Glu-activated astrocytes (P < 0.05). SSa at 2.5 mg/L showed the strongest inhibitory effects against astrocyte activation and maintained nearly normal level of astrocyte activation in comparison with the control group (P > 0.05).

CONCLUSIONSGlu-induced activation of rat hippocampal astrocytes can be inhibited by SSa, whose antiepileptic effects is probably mediated by inhibition of hippocampal astrocyte activation.

Animals ; Animals, Newborn ; Astrocytes ; cytology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Female ; Glial Fibrillary Acidic Protein ; biosynthesis ; Glutamic Acid ; pharmacology ; Hippocampus ; cytology ; Male ; Oleanolic Acid ; analogs & derivatives ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Saponins ; pharmacology

OBJECTIVETo study the effect of saikosaponins, the active ingredients of Bupleurum chinense DC. on glutamate and GABA expressions in the hippocampus of slow kindling rats induced by pentetrazole.

METHODSForty-eight healthy Sprague-Dawley rats were randomly divided into 6 equal groups, namely the blank control group (group A), normal saline (NS) group (group B), sodium valproate group (group C), and 3 saikosaponins groups of high, medium and small doses (groups D, E, and F, respectively). The rats in each group other than group A were given corresponding treatments after slow kindling by pentetrazole. After 4 weeks of treatment, the rats were sacrifices and the brain tissues were sampled, sliced and stained by immunohistochemically, and the results were analyzed according to the positive cell number and gray scale.

RESULTSIn CA1 region, the glutamate-positive cell number and gray scale of group B was significantly different from the other groups (P<0.05), but such difference was not observed in the CA2 and DG (P>0.05); In CA1, CA2 and DG of the hippocampus, the GABA-positive cell number of group B was significantly greater but the gray scale lower than those of the other groups (P<0.05). In CA1 and CA2 regions of the hippocampus, the glutamate- and GABA-positive cell ratio of group B was significantly lower than that of the other groups (P<0.05), but in CA1, CA2, and DG region of the hippocampus, the ratio of gray scale between glutamate- and GABA-positive cells was comparable between the groups (P>0.05).

CONCLUSIONThe expression of glutamate and GABA, especially the latter, increased in chronic kindling rat hippocampus. Saikosaponins intervene in such changes of glutamate and GABA to contain their expressions within normal range, which may be one of the mechanisms of saikosaponins to inhibit slow kindling induced by pentetrazole.

Animals ; Female ; Glutamic Acid ; biosynthesis ; Hippocampus ; drug effects ; metabolism ; Immunohistochemistry ; Kindling, Neurologic ; metabolism ; Male ; Oleanolic Acid ; analogs & derivatives ; pharmacology ; Pentylenetetrazole ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Saponins ; pharmacology ; gamma-Aminobutyric Acid ; biosynthesis

OBJECTIVETo investigate the uppressive effects of par-4 antisense oligodeoxynucleotide on the up-regulation of intracellular calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects.

METHODSCationic lipid-mediated par-4 antisense oligodeoxynucleotide (par-4-AS-ODN) was transfected into PC12 cells and followed by glutamate for treatment. Mismatch oligodeoxy-nucleotide (MS-ODN) was used as the control. Morphological assessment and evaluation of the anti-apoptosis effects of par-4-AS-ODN on PC12 cells were performed by laser scanning confocal microscopy by double staining of the cells with Hoechest 33258/propidium iodide (Hoe/PI) and flow cytometry respectively. The mRNA and protein levels of calpain 10 and par-4 were measured by RT-PCR and Western blot. Intracellular calcium concentration was determined by using laser scanning confocal microscope with Fura-2/AM as the fluorescent dye.

RESULTSPar-4-AS-ODN repress the increase of par-4 protein in PC12 cell (52.3 +/- 5.0 vs 90.0 +/- 3.2, < 0.01). Par-4-AS-ODN significantly inhibited the apoptosis of PC12 cells induced by glutamate (53% vs 31%, < 0.01). Par-4-AS-ODN significantly suppress the up-regulation of intracellular calcium concentration in PC12 cells induced by glutamate (Rate of fluorescent density: 167.9 +/- 32.4 vs 228.8 +/- 36.8, < 0.01). Par-4-AS-ODN inhibited the increase of calpain 10 mRNA in PC12 cells induced by glutamate (46.3 +/- 3.7 vs 34.8 +/- 2.1, < 0.01).

CONCLUSIONSpar-4-AS-ODN enables to inhibit apoptosis of PC12 cells induced by glutamate. The mechanism of the inhibition may be closely related to suppression of the up-regulation of intracellular calcium concentration and calpain transcription expression.

Animals ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; biosynthesis ; genetics ; Calcium ; metabolism ; Calpain ; biosynthesis ; genetics ; Glutamic Acid ; pharmacology ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; PC12 Cells ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Transfection ; Up-Regulation

The glyoxylate cycle was hypothesed to be indispensable for glutamate overproduction in coryneform bacteria, for it was thought to fulfill anaplerotic functions and to supply energy during the growth phase. During glutamate overproduction phase, however, it has been noted that the high level of the cycle is detrimental to the glutamate production. In order to clarify the relationship between the glutamate production and the glyoxylate cycle, a chromosomal aceA-disrupted mutant of wild-type C. glutamicum ATCC 13032 was constructed. The isocitrate lyase (ICL) activity of the parental strain was 0.011 u/mg of protein and reached 1.980 u/mg of protein after acetate induction; the mutant strain WTdeltaA, however, had no detectable ICL activity and was no longer able to grow on minimal medium with acetate as the sole carbon source. Compared with the wild-type C. glutamicum WT, the mutant strain WTdeltaA, exhibited the same growth rate with glucose as the sole carbon source, indicating glyoxylate cycle is not required for its growth on glucose. On the contrary, the glutamate production in WTdeltaA was severely impaired and more residual glucose was found in the fermentation broth at the end of fermentation with the mutant strain than with the wild-type strain. Further investigations into the relationship between the glutamate production and the glyoxylate cycle are under the way, which may help to elucidate the mechanism of glutamate overproduction.
Corynebacterium glutamicum ; genetics ; growth & development ; metabolism ; Culture Media ; Fermentation ; Glucose ; metabolism ; Glutamic Acid ; biosynthesis ; Glyoxylates ; metabolism ; Isocitrate Lyase ; metabolism

To investigate the effect of propofol on the release of glutamate and gamma-aminobutyric acid (GABA) from rat hippocampal synatosomes, synaptosomes was made from hippocampus and incubated with artificial cerebrospinal fluid (aCSF). With the experiment of Ca(2+)-dependent release of glutamate and GABA, dihydrokainic acid (DHK) and nipectic acid were added into aCSF. For the observation of Ca(2+)-independent release of glutamate and GABA, no DHK, nipectic acid and Ca2+ were added from aCSF. The release of glutamate and GABA were evoked by 20 micromol/L veratridine or 30 mmol/L KCI. The concentration of glutamate and GABA in aCSF was measured by using high-performance liquid chromatography (HPLC). 30, 100 and 300 micromol/L propofol significantly inhibited veratridine-evoked Ca(2+)-dependent release of glutamate and GABA (P < 0.01 or P < 0. 05). However, propofol showed no effect on elevated KCl-evoked Ca(2+)-dependent release of glutamate and GABA (P > 0.05). Veratridine or elevated KCI evoked Ca(2+)-independent release of glutamate and GABA was not affected significantly by propofol (P > 0.05). Propofol could inhibit Ca(2+)-dependent release of glutamate and GABA. However, it has no effect on the Ca(2+)-independent release of glutamate and GABA.
Anesthetics, Intravenous/pharmacology ; Calcium/metabolism ; Glutamic Acid/*biosynthesis ; Hippocampus/*metabolism ; Propofol/*pharmacology ; Rats, Sprague-Dawley ; Synaptosomes/*metabolism ; gamma-Aminobutyric Acid/*biosynthesis

To explore the mechanism of interleukin-1beta (IL-1beta) in the onset of seizure and the effect of IL-1beta on the expression of adenylyl cyclase (AC) in rats with seizure induced by L-glutamate. Experimental rats were first injected with IL-1beta and then L-glutamate (a dose under the threshold) was injected into the right lateral ventricle. The rats were sacrificed 4 h after the onset of epileptic activity and examined for changes in behavior, immunohistochemistry and compared with those with seizure induced by L-glutamate alone. It was found that the expression of AC in hippocampal and neocortex of rats with seizure induced by IL-1beta and L-glutamate were stronger than that of control group (P<0.05), without significant difference found between the L-glutamate group and IL-1beta plus L-glutamate group in the expression of AC, the latent period and the severity of seizure. When IL-ra were given (i.c.v.) first, there was no epileptic activity and the expression of AC did not increase. There were no differences in the expression of AC of rats with IL-1ra and that of control rats. But when 2-methyl-2-(carboxycyclopropyl) glycine (MCCG) was given (i.c.v.) first, the strongest expression of AC, the shortest latent period and the the most serious seizure activities were observed. The results indicated that IL-1beta could facilitate the onset of epilepsy induced by L-glutamate through IL-1R, metabotropic glutamate receptors might work with IL-1R and the increased expression of AC might be involved in the process.
Adenylyl Cyclases ; biosynthesis ; genetics ; Animals ; Glutamic Acid ; Hippocampus ; metabolism ; Interleukin-1 ; pharmacology ; Male ; Neocortex ; metabolism ; Rats ; Seizures ; chemically induced ; enzymology

The antisense approach and RT-PCR were used to study the effects of muscarinic receptors on the scores of morphine-withdrawal syndrome and the expression of NMDA receptor subtypes (NR(1A) and NR(2A)) mRNA in rat spinal cord and brainstem. The concentrations of glutamate in periaqueductal grey (PAG) of morphine-withdrawal rats were determined by capillary electrophoresis with laser-induced fluorescence detection. The data showed that the NR(1A) and NR(2A) mRNA levels were increased significantly in the spinal cord and brainstem 1 h after the injection of naloxone (4 mg/kg, i.p.) in morphine-dependent rats. Moreover, in morphine-dependent rats pretreated (i.p.) with scopolamine (0.5 mg/kg), or pirenzepine (10 mg/kg), MK801 (0.125 mg/kg), L-N-nitroarginine methylester (10 mg/kg) 30 min before naloxone injection, the NR(1A) and NR(2A) mRNA levels were significantly lower than those of 1 h morphine-withdrawal rats. Intrathecal injection of NR(1A) or M(2) receptor antisense oligonucleotides (A-oligo, 4 microg/per rat) 24 h prior to naloxone challenge could block the morphine withdrawal symptoms including wet dog shaking, irritability, salivation, diarrhea, chewing and weight loss. Meanwhile, in morphine-dependent rats the NR(1A) mRNA levels in the spinal cord and brainstem were down-regulated by intrathecal injection of M(2) receptor A-oligo. The glutamate concentrations in PAG microdialysis were increased to a maximal level 15 min after naloxone injection. The glutamate response was inhibited by pretreatment with M(2) receptor A-oligo but not by M(1) A-oligo. The results suggest that the expression of NMDA receptors and the release of glutamate in brainstem are involved in the processes of morphine withdrawal and that the NMDA receptor expression is possibly regulated by the muscarinic receptors during morphine withdrawal.
Animals ; Brain Stem ; metabolism ; Glutamic Acid ; metabolism ; Male ; Morphine ; adverse effects ; Periaqueductal Gray ; metabolism ; physiology ; Rats ; Rats, Sprague-Dawley ; Receptors, Muscarinic ; physiology ; Receptors, N-Methyl-D-Aspartate ; biosynthesis ; genetics ; Spinal Cord ; metabolism ; Substance Withdrawal Syndrome ; genetics ; metabolism