Efficient production of γ-aminobutyric acid using engineered Escherichia coli whole-cell catalyst.

γ-Aminobutyric acid (GABA) has been widely used in the food, feed, pharmaceutical, and chemical industry fields. Previously, we developed a whole-cell catalyst capable of converting L-glutamate (L-Glu) into GABA by overexpressing the glutamate decarboxylase gene (gadz11) from Bacillus sp. Z11 in Escherichia coli BL21(DE3). However, to enhance cell permeability, a freeze-thaw treatment is required, and to enhance GADZ11 activity, pyridoxal 5'-phosphate (PLP) must be added to the reaction system. The aim of this study is to provide a more efficient approach for GABA production by engineering the recombinant E. coli above. First, the inducible expression conditions of the gadz11 in E. coli were optimized to 37 °C for 6 h. Next, an ideal engineered strain was produced via increasing cell permeability by overexpressing sulA and eliminating PLP dependence by constructing a self-sufficient system. Furthermore, an efficient whole-cell biocatalytic process was optimized. The optimal substrate concentration, cell density, and reaction temperature were 1.0 mol/L (the molecular ratio of L-Glu to L-monosodium glutamate (L-MSG) was 4:1), 15 and 37 °C, respectively. Finally, a whole-cell bioconversion procedure was performed in a 3-L bioreactor under optimal conditions. The strain could be reused for at least two cycles with GABA yield, productivity and conversion ratio of 206.2 g/L, 117.8 g/L/h and 100.0%, respectively. This is currently the highest GABA productivity from a mixture of L-Glu and L-MSG reported without the addition of cofactors or additional treatment of cells. This work demonstrates that the novel engineered E. coli strain has the potential for application in large-scale industrial GABA production.

Glutamate decarboxylase-1 is essential for efficient acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation.

Glutamate decarboxylase (GAD) is a Ca2+ -calmodulin-activated, cytosolic enzyme that produces γ-aminobutyrate (GABA) as the committed step of the GABA shunt. This pathway bypasses the 2-oxoglutarate to succinate reactions of the tricarboxylic acid (TCA) cycle. GABA also accumulates during many plant stresses. We tested the hypothesis that AtGAD1 (At5G17330) facilitates Arabidopsis acclimation to Pi deprivation. Quantitative RT-PCR and immunoblotting revealed that AtGAD1 transcript and protein expression is primarily root-specific, but inducible at lower levels in shoots of Pi-deprived (-Pi) plants. Pi deprivation reduced levels of the 2-oxoglutarate dehydrogenase (2-OGDH) cofactor thiamine diphosphate (ThDP) in shoots and roots by > 50%. Growth of -Pi atgad1 T-DNA mutants was significantly attenuated relative to wild-type plants. This was accompanied by: (i) an > 60% increase in shoot and root GABA levels of -Pi wild-type, but not atgad1 plants, and (ii) markedly elevated anthocyanin and reduced free and total Pi levels in leaves of -Pi atgad1 plants. Treatment with 10 mM GABA reversed the deleterious development of -Pi atgad1 plants. Our results indicate that AtGAD1 mediates GABA shunt upregulation during Pi deprivation. This bypass is hypothesized to circumvent ThDP-limited 2-OGDH activity to facilitate TCA cycle flux and respiration by -Pi Arabidopsis.

Postnatal development of the hippocampal GABAergic system in rats genetically prone to audiogenic seizures.

Epileptogenesis can be associated with altered genetic control of the GABAergic system. Here we analyzed Krushinsky-Molodkina (KM) rats genetically prone to audiogenic epilepsy. KM rats express fully formed audiogenic seizures (AGSs) not early, then they reach 3 months. At the age of 1-2 months, KM rats either do not express AGS or demonstrate an incomplete pattern of seizure. Such long-term development of AGS susceptibility makes KM rats an especially convenient model to investigate the mechanisms and dynamics of the development of inherited epilepsy. The analysis of the GABAergic system of the hippocampus of KM rats was done during postnatal development at the 15th, 60th, and 120th postnatal days. Wistar rats of corresponding ages were used as a control. In the hippocampus of KM pups, we observed a decrease in the expression of glutamic acid decarboxylase 67 (GAD67) and parvalbumin (PV), which points to a decrease in the activity of GABAergic neurons. Analysis of the 2-month-old KM rats showed an increase in GAD67 and PV expression while synapsin I and vesicular GABA transporter (VGAT) were decreased. In adult KM rats, the expression of GAD67, PV, and synapsin I was upregulated. Altogether, the obtained data indicate significant alterations in GABAergic transmission in the hippocampus of audiogenic KM rats during the first postnatal months.

Age-related changes in glutamic acid decarboxylase 1 gene expression in the medial prefrontal cortex and ventral hippocampus of fear-potentiated rats subjected to isolation stress.

Gamma-aminobutyric acid (GABA) plays a crucial role as a neurotransmitter in anxiety circuits, prominently in the hippocampus, amygdala, and prefrontal cortex. The synthesis of GABA in the central nervous system is primarily governed by glutamic acid decarboxylase 67 (GAD67). Aging is associated with emotional alterations, and isolation stress has been linked to increased anxiety. This study aimed to investigate the impact of aging on the gene expression of GAD67 (Gad1) in the medial prefrontal cortex (m PC) and ventral hippocampus (v Hip) of fear-potentiated rats subjected to isolation stress. To conduct the study, Wistar rats of different age groups 21-day-old (immature), 42-day-old (peri-adolescent), and 365-day-old (mature adult) were utilized. Each age level was categorized into four groups: 1) Control group - no pre-stressor, no maze, no drug, 2) Innate fear group (M) - no pre-stressor, maze, no drug, 3) Fear-potentiated group (IM) - isolation pre-stressor for 120 min, maze, no drug, and 4) Diazepam-treated group (IMD) - isolation pre-stressor for 120 min, maze, and diazepam administration. Following the tests, the (m PC) and (v Hip) regions were dissected, and Gad1 gene expression changes were assessed using Real-time PCR technique. The results revealed that, across all age groups, Gad1 expression in both the (m PC) and (v Hip) was significantly higher in the fear-potentiated groups (IM) compared to the control and innate fear (M) groups. Notably, in aged 365-day-old rats from the innate fear group (M), the expression of Gad1 in (v Hip) was also higher than that in the control group. Additionally, aged fear-potentiated rats exhibited elevated Gad1 gene expression in both structures compared to other age groups. These findings suggest that isolation stress before exposure to the elevated plus maze (EPM) can elevate Gad1 gene expression in both the (v Hip) and (m PC), and age may play a role in modulating its expression.

Two putative glutamate decarboxylases of Streptococcus pneumoniae as possible antigens for the production of anti-GAD65 antibodies leading to type 1 diabetes mellitus / Dos supuestas glutamato descarboxilasas de Streptococcus pneumoniae como posibles antígenos para la producción de anticuerpos anti-GAD65 que conducen a la diabetes mellitus tipo 1

Type 1 diabetes mellitus (T1DM) has been increasing in prevalence in the last decades and has become a global burden. Autoantibodies against human glutamate decarboxylase (GAD65) are among the first to be detected at the onset of T1DM. Diverse viruses have been proposed to be involved in the triggering of T1DM because of molecular mimicry, i.e., similarity between parts of some viral proteins and one or more epitopes of GAD65. However, the possibility that bacterial proteins might also be responsible for GAD65 mimicry has been seldom investigated. To date, many genomes of Streptococcus pneumoniae (the pneumococcus), a prominent human pathogen particularly prevalent among children and the elderly, have been sequenced. A dataset of more than 9000 pneumococcal genomes was mined and two different (albeit related) genes (gadA and gadB), presumably encoding two glutamate decarboxylases similar to GAD65, were found. The various gadASpn alleles were present only in serotype 3 pneumococci belonging to the global lineage GPSC83, although some homologs have also been discovered in two subspecies of Streptococcus constellatus (pharyngis and viborgensis), an isolate of the group B streptococci, and several strains of Lactobacillus delbrueckii. Besides, gadBSpn alleles are present in > 10% of the isolates in our dataset and represent 16 GPSCs with 123 sequence types and 20 different serotypes. Sequence analyses indicated that gadA- and gadB-like genes have been mobilized among different bacteria either by prophage(s) or by integrative and conjugative element(s), respectively. Substantial similarities appear to exist between the putative pneumococcal glutamate decarboxylases and well-known epitopes of GAD65. In this sense, the use of broader pneumococcal conjugate vaccines such as PCV20 would prevent the majority of serotypes expressing those genes that might potentially contribute to T1DM.(AU)

[Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum].

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 µmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Two putative glutamate decarboxylases of Streptococcus pneumoniae as possible antigens for the production of anti-GAD65 antibodies leading to type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) has been increasing in prevalence in the last decades and has become a global burden. Autoantibodies against human glutamate decarboxylase (GAD65) are among the first to be detected at the onset of T1DM. Diverse viruses have been proposed to be involved in the triggering of T1DM because of molecular mimicry, i.e., similarity between parts of some viral proteins and one or more epitopes of GAD65. However, the possibility that bacterial proteins might also be responsible for GAD65 mimicry has been seldom investigated. To date, many genomes of Streptococcus pneumoniae (the pneumococcus), a prominent human pathogen particularly prevalent among children and the elderly, have been sequenced. A dataset of more than 9000 pneumococcal genomes was mined and two different (albeit related) genes (gadA and gadB), presumably encoding two glutamate decarboxylases similar to GAD65, were found. The various gadASpn alleles were present only in serotype 3 pneumococci belonging to the global lineage GPSC83, although some homologs have also been discovered in two subspecies of Streptococcus constellatus (pharyngis and viborgensis), an isolate of the group B streptococci, and several strains of Lactobacillus delbrueckii. Besides, gadBSpn alleles are present in > 10% of the isolates in our dataset and represent 16 GPSCs with 123 sequence types and 20 different serotypes. Sequence analyses indicated that gadA- and gadB-like genes have been mobilized among different bacteria either by prophage(s) or by integrative and conjugative element(s), respectively. Substantial similarities appear to exist between the putative pneumococcal glutamate decarboxylases and well-known epitopes of GAD65. In this sense, the use of broader pneumococcal conjugate vaccines such as PCV20 would prevent the majority of serotypes expressing those genes that might potentially contribute to T1DM. These results deserve upcoming studies on the possible involvement of S. pneumoniae in the etiopathogenesis and clinical onset of T1DM.

Sex differences in anxiety and threat avoidance in GAD65 knock-out mice.

Anxiety disorders have been linked to a disbalance of excitation and inhibition in a network of brain structures comprising frontal cortical regions, the amygdala and the hippocampus, among others. Recent imaging studies suggest sex differences in the activation of this anxiety network during the processing of emotional information. Rodent models with genetically altered ϒ-amino butyric acid (GABA) neurotransmission allow studying the neuronal basis of such activation shifts and their relation to anxiety endophenotypes, but to date sex effects have rarely been addressed. Using mice with a null mutation of the GABA synthetizing enzyme glutamate decarboxylase 65 (GAD65-/-), we started to compare anxiety-like behavior and avoidance in male vs. female GAD65-/- mice and their wildtype littermates. In an open field, female GAD65-/- mice displayed increased activity, while male GAD65-/- mice showed an increased adaptation of anxiety-like behavior over time. GAD65-/- mice of both sexes had a higher preference for social interaction partners, which was further heightened in male mice. In male mice higher escape responses were observed during an active avoidance task. Together, female mice showed more stable emotional responses despite GAD65 deficiency. To gain insights into interneuron function in network structures controlling anxiety and threat perception, fast oscillations (10-45 Hz) were measured in ex vivo slice preparations of the anterior cingulate cortex (ACC). GAD65-/- mice of both sexes displayed increased gamma power in the ACC and a higher density of PV-positive interneurons, which are crucial for generating such rhythmic activity. In addition, GAD65-/- mice had lower numbers of somatostatin-positive interneurons in the basolateral amygdala and in the dorsal dentate gyrus especially in male mice, two key regions important for anxiety and active avoidance responses. Our data suggest sex differences in the configuration of GABAergic interneurons in a cortico-amygdala-hippocampal network controlling network activity patterns, anxiety and threat avoidance behavior.

GAD65 deficient mice are susceptible to ethanol-induced impairment of motor coordination and facilitation of cerebellar neuronal firing.

γ-aminobutyric acid (GABA) is a major inhibitory neurotransmitter and its concentrations in the brain could be associated with EtOH-induced impairment of motor coordination. GABA is synthesized by two isoforms of glutamate decarboxylase (GAD): GAD65 and GAD67. Mice deficient in GAD65 (GAD65-KO) can grow up to adulthood, and show that GABA concentration in their adult brains was 50-75% that of wild-type C57BL/6 mice (WT). Although a previous study showed that there was no difference in recovery from the motor-incoordination effect of acute intraperitoneally administered injections of 2.0 g/kg EtOH between WT and GAD65-KO, the sensitivity of GAD65-KO to acute EtOH-induced ataxia has not been fully understood. Here, we sought to determine whether motor coordination and spontaneous firing of cerebellar Purkinje cells (PCs) in GAD65-KO are more sensitive to the effect of EtOH than in WT. Motor performance in WT and GAD65-KO was examined by rotarod and open-field tests following acute administration of EtOH at lower-doses, 0.8, 1.2 and 1.6 g/kg. In a rotarod test, there was no significant difference between WT and GAD65-KO in terms of baseline motor coordination. However, only the KO mice showed a significant decrease in rotarod performance of 1.2 g/kg EtOH. In the open-field test, GAD65-KO showed a significant increase in locomotor activity after 1.2 and 1.6 g/kg EtOH injections, but not WT. In in vitro studies of cerebellar slices, the firing rate of PCs was increased by 50 mM EtOH in GAD65-KO compared with WT, whereas no difference was observed in the effect of EtOH at more than 100 mM between the genotypes. Taken together, GAD65-KO are more susceptible to the effect of acute EtOH exposure on motor coordination and PC firing than WT. This different sensitivity could be attributed to the basal low GABA concentration in the brain of GAD65-KO.

Continuous production of gamma aminobutyric acid by engineered and immobilized Escherichia coli whole-cells in a small-scale reactor system.

γ-Amino butyric acid (GABA) is a non-proteinogenic amino acid and a human neurotransmitter. Recently, increasing demand for food additives and biodegradable bioplastic monomers, such as nylon 4, has been reported. Consequently, considerable efforts have been made to produce GABA through fermentation and bioconversion. To realize bioconversion, wild-type or recombinant strains harboring glutamate decarboxylase were paired with the cheap starting material monosodium glutamate, resulting in less by-product formation and faster production compared to fermentation. To increase the reusability and stability of whole-cell production systems, this study used an immobilization and continuous production system with a small-scale continuous reactor for gram-scale production. The cation type, alginate concentration, barium concentration, and whole-cell concentration in the beads were optimized and this optimization resulted in more than 95 % conversion of 600 mM monosodium glutamate to GABA in 3 h and reuse of the immobilized cells 15 times, whereas free cells lost all activity after the ninth reaction. When a continuous production system was applied after optimizing the buffer concentration, substrate concentration, and flow rate, 165 g of GABA was produced after 96 h of continuous operation in a 14-mL scale reactor. Our work demonstrates the efficient and economical production of GABA by immobilization and continuous production in a small-scale reactor.

Localization and Diagnostic Specificity of Glutamic Acid Decarboxylase Transcript Alterations in the Dorsolateral Prefrontal Cortex in Schizophrenia.

BACKGROUND: Working memory (WM) deficits in schizophrenia are thought to reflect altered inhibition in the dorsolateral prefrontal cortex (DLPFC). This interpretation is supported by findings of lower transcript levels of the 2 enzymes, GAD67 and GAD65, which mediate basal and activity-dependent GABA (gamma-aminobutyric acid) synthesis, respectively. However, the relative magnitude, location within the depth of the DLPFC, and specificity to the disease process of schizophrenia of alterations in GAD67 and/or GAD65 remain unclear. METHODS: Levels of GAD67 and GAD65 messenger RNAs (mRNAs) in superficial (layers 2/superficial 3) and deep (deep layer 6/white matter) zones of the DLPFC were quantified by quantitative polymerase chain reaction in subjects with schizophrenia (n = 41), major depression (n = 42), or bipolar disorder (n = 39) and unaffected comparison (n = 43) subjects. RESULTS: Relative to the unaffected comparison group, GAD67 and GAD65 mRNA levels in the schizophrenia group were lower (p = .039, effect size = -0.69 and p = .027, effect size = -0.72, respectively) in the superficial zone but were unaltered in the deep zone. In the major depression group, only GAD67 mRNA levels were lower and only in the superficial zone (p = .089, effect size = 0.70). No differences were detected in the bipolar disorder group. Neither GAD67 nor GAD65 mRNA alterations were explained by psychosis, mood disturbance, or common comorbid factors. CONCLUSIONS: Alterations in markers of GABA synthesis demonstrated transcript, DLPFC zone, and diagnostic specificity. Given the dependence of WM on GABA neurotransmission in the superficial DLPFC, our findings suggest that limitations to GABA synthesis in this location contribute to WM impairments in schizophrenia, especially during demanding WM tasks, when GABA synthesis requires the activity of both GAD67 and GAD65.

Integrating Enzyme Evolution and Metabolic Engineering to Improve the Productivity of Γ-Aminobutyric Acid by Whole-Cell Biosynthesis in Escherichia Coli.

γ-Aminobutyric acid (GABA) is used widely in various fields, such as agriculture, food, pharmaceuticals, and biobased chemicals. Based on glutamate decarboxylase (GadBM4) derived from our previous work, three mutants, GadM4-2, GadM4-8, and GadM4-31, were obtained by integrating enzyme evolution and high-throughput screening methods. The GABA productivity obtained through whole-cell bioconversion using recombinant Escherichia coli cells harboring mutant GadBM4-2 was enhanced by 20.27% compared to that of the original GadBM4. Further introduction of the central regulator GadE of the acid resistance system and the enzymes from the deoxyxylulose-5-phosphate-independent pyridoxal 5'-phosphate biosynthesis pathway resulted in a 24.92% improvement in GABA productivity, reaching 76.70 g/L/h without any cofactor addition with a greater than 99% conversion ratio. Finally, when one-step bioconversion was applied for the whole-cell catalysis in a 5 L bioreactor, the titer of GABA reached 307.5 ± 5.94 g/L with a productivity of 61.49 g/L/h by using crude l-glutamic acid (l-Glu) as the substrate. Thus, the biocatalyst constructed above combined with the whole-cell bioconversion method represents an effective approach for industrial GABA production.

GadR4 mediates the acid resistance and pathogenicity of Listeria monocytogenes 10403S by negatively regulating the gadT2/gadD2 cluster.

Listeria monocytogenes is an important foodborne pathogen that can survive under acidic conditions. The glutamate decarboxylase (GAD) system is one of the acid resistance systems of L. monocytogenes. It usually comprises two glutamate transporters (GadT1/T2) and three glutamate decarboxylases (GadD1/D2/D3). Among them, gadT2/gadD2 contributes most significantly to the acid resistance of L. monocytogenes. However, the regulation mechanisms of gadT2/gadD2 still remain unclear. The results of this study indicated that gadT2/gadD2 deletion significantly decreases the survival rate of L. monocytogenes under different acidic conditions, including brain and heart infusion (BHI) broth, with a pH of 2.5, 2% citric acid, 2% acetic acid and 2% lactic acid. Further, gadT2/gadD2 cluster was expressed in the representative strains in response to alkaline stress rather than acid stress. To explore the regulation of gadT2/gadD2, we knocked out the five transcriptional factors belonging to the Rgg family in L. monocytogenes 10403S. We found that the deletion of gadR4, which exhibits the highest homology with the gadR of Lactococcus lactis, resulted in a significant increase in the survival rate of L. monocytogenes upon acid stress. Western blot analysis showed that gadR4 deletion significantly increased the gadD2 expression of L. monocytogenes under alkaline and neutral conditions. Furthermore, the gfp reporter gene showed that gadR4 deletion significantly increased the expression of the gadT2/gadD2 cluster. Adhesion and invasion assays indicated that gadR4 deletion significantly increased the rates of adhesion and invasion of L. monocytogenes to epithelial Caco-2 cells. Virulence assays showed that gadR4 knockout significantly improved the colonization ability of L. monocytogenes in the livers and spleens of the infected mice. Taken together, our results showed that GadR4, a transcription factor belonging to the Rgg family, negatively regulates the gadT2/gadD2 cluster, thus, reducing the acid stress tolerance and pathogenicity of L. monocytogens 10403S. Our results provide a better understanding of the regulation of the GAD system of L. monocytogenes and a novel approach to potentially prevent and control listeriosis.

Effect of RNA interference with glutamate decarboxylase on acid resistance of Trichinella spiralis.

Trichinella spiralis is a zoonotic parasite that infects most mammals, even humans. Glutamate decarboxylase (GAD) is an important enzyme in glutamate-dependent acid resistance system 2 (AR2), but the GAD of T. spiralis in AR2 is unclear. We aimed to investigate the role of T. spiralis glutamate decarboxylase (TsGAD) in AR2. We silenced the TsGAD gene to evaluate the AR of T. spiralis muscle larvae (ML) in vivo and in vitro via siRNA. The results showed that recombinant TsGAD was recognized by anti-rTsGAD polyclonal antibody (57 kDa), and qPCR indicated that TsGAD transcription peaked at pH 2.5 for 1 h compared to that with pH 6.6 phosphate-buffered saline. Indirect immunofluorescence assays revealed that TsGAD was expressed in the epidermis of ML. After TsGAD silencing in vitro, TsGAD transcription and the survival rate of ML decreased by 15.2% and 17%, respectively, compared with those of the PBS group. Both TsGAD enzymatic activity and the acid adjustment of siRNA1-silenced ML were weakened. In vivo, each mouse was orally infected with 300 siRNA1-silenced ML. On days 7 and 42 post-infection, the reduction rates of adult worms and ML were 31.5% and 49.05%, respectively. Additionally, the reproductive capacity index and larvae per gram of ML were 62.51±7.32 and 1250.22±146.48, respectively, lower than those of the PBS group. Haematoxylin-eosin staining revealed many inflammatory cells infiltrating the nurse cells in the diaphragm of mice infected with siRNA1-silenced ML. The survival rate of the F1 generation ML was 27% higher than that of the F0 generation ML, but there was no difference from the PBS group. These results first indicated that GAD plays a crucial role in AR2 of T. spiralis. TsGAD gene silencing reduced the worm burden in mice, providing data for the comprehensive study of the AR system of T. spiralis and a new idea for the prevention of trichinosis.

Parvalbumin in the metabolic pathway of glutamate and γ-aminobutyric acid: Influence on expression of GAD65 and GAD67.

Parvalbumin-expressing neurons are a type of inhibitory intermediate neuron that play an important role in terminating seizures. The aim of the present study was to use lentiviral construction and packaging technology to overexpress and silence the parvalbumin gene in pheochromocytoma (PC12) cells, and to evaluate how parvalbumin influences the metabolic pathway involving glutamate and γ-aminobutyric acid (GABA). In this work, Immunofluorescence staining was used to verify the differentiation of PC12 cells into neurons after adding nerve growth factor (NGF). Western blotting and real-time quantitative polymerase chain reaction (qRT-PCR) were used to confirm lentivirus-mediated knockdown or overexpression of parvalbumin. Expression of parvalbumin, the 65-kDa GAD isoform (GAD65), and the 67-kDa GAD isoform (GAD67) in neuronal cells was examined at the mRNA and protein levels using qRT-PCR, western blotting and immunofluorescence staining, while intracellular glutamate and GABA levels were determined by high performance liquid chromatography (HPLC). We demonstrate that the expression of parvalbumin is associated with GAD65 and GAD67. Interestingly, overexpression of parvalbumin up-regulated GAD65 and GAD67, increased GABA concentration, and decreased glutamate concentration. Silencing of parvalbumin led to the opposite effects. Altogether, parvalbumin affected the expression of GAD65 and GAD67, thereby influencing the metabolic pathway involving glutamate and GABA.

Coordinated regulation of Bacteroides thetaiotaomicron glutamate decarboxylase activity by multiple elements under different pH.

Glutamate decarboxylase catalyzes the conversion of glutamate to γ-aminobutyric acid, which plays a vital role in the gut-brain axis. Herein, a novel glutamate decarboxylase from Bacteroides thetaiotaomicron (BTGAD) was heterologously expressed. BTGAD possessed high catalytic efficiency at 60℃ and pH 3.6. As pH response, N-terminal sequence (NTS), C-terminal sequence (CTS), and ß-hairpin in BTGAD coordinately regulated its activity under different pH. NTS folded into a loop under acidic pH, and the truncation of NTS severely reduced its activity to 4.2%. While CTS occupied the active site under neutral pH and became disordered to release the inhibition effect under acidic conditions. The ß-hairpin, located near the active site, swung and formed open and closed conformations, which acted as an activity switch. This study provides the molecular basis for the coordinated regulation mechanism of BTGAD and lays a theoretical foundation for understanding the metabolism of dietary glutamate and its interaction relationships with the gut-brain axis.

Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum / 生物工程学报

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Expression of GAD65/67 and VGLUT2 in Mediobasal Nuclei of Rat Hypothalamus during Aging.

The expression of glutamate decarboxylase GAD65/67, an enzyme of GABA synthesis, and vesicular glutamate transporter 2 (VGLUT2) in the arcuate, dorsomedial, and ventromedial nuclei of the hypothalamus of young (3 months), adult (12 months), and old male rats (24 months) was studied by Western blotting. In old rats, an increase in the expression of GAD65/67 in the arcuate and dorsomedial, VGLUT2 in the arcuate, dorsomedial, and ventromedial nuclei was observed. Thus, an increase in opposite processes of inhibition and excitation is observed in the hypothalamic nuclei during aging.

The upregulation of glutamate decarboxylase 67 against hippocampal excitability damage in male fetal rats by prenatal caffeine exposure.

As a kind of xanthine alkaloid, caffeine is widely present in beverages, food, and analgesic drugs. Our previous studies have shown that prenatal caffeine exposure (PCE) can induce programmed hypersensitivity of the hypothalamic-pituitary-adrenal (HPA) axis in offspring rats, which is involved in developing many chronic adult diseases. The present study further examined the potential molecular mechanism and toxicity targets of hippocampal dysfunction, which might mediate the programmed hypersensitivity of the HPA axis in offspring. Pregnant rats were intragastrically administered with 0, 30, and 120 mg/kg/day caffeine from gestational days (GD) 9-20, and the fetal rats were extracted at GD20. Rat fetal hippocampal H19-7/IGF1R cell line was treated with caffeine, adenosine A2A receptor (A2AR) agonist (CGS-21680) or adenylate cyclase agonist (forskolin) plus caffeine. Compared with the control group, hippocampal neurons of male fetal rats by PCE displayed increased apoptosis and reduced synaptic plasticity, whereas glutamate decarboxylase 67 (GAD67) expression was increased. Moreover, the expression of A2AR was down-regulated, PCE inhibited the cAMP/PKA/CREB/BDNF/TrkB pathway. Furthermore, the results in vitro were consistent with the in vivo study. Both CGS21680 and forskolin could reverse the above alteration caused by caffeine. These results indicated that PCE inhibits the BDNF pathway and mediates the hippocampus's glutamate (Glu) excitotoxicity. The compensatory up-regulation of GAD67 unbalanced the Glu/gamma-aminobutyric acid (GABA)ergic output, leading to the impaired negative feedback to the hypothalamus and hypersensitivity of the HPA axis.

R2R3-MYB transcription factor FaMYB5 is involved in citric acid metabolism in strawberry fruits.

The citrate content of strawberry fruits affects their organoleptic quality. However, little is known about the transcriptional regulatory mechanisms of citric acid metabolism in strawberry fruits. In this study, the R2R3-MYB transcription factor FaMYB5 was identified and placed in the R2R3-MYB subfamily. FaMYB5 is found in the nucleus and shows tissue- and stage-specific expression levels. Citric acid content was positively correlated with FaMYB5 transcript levels. Upregulated FaMYB5 increased citric acid accumulation in transient FaMYB5-overexpressing strawberry fruits, whereas transient RNA silencing of FaMYB5 in strawberry fruits resulted in a reduction of citric acid content. The role of FaMYB5 was verified using stable transgenic NC89 tobacco. Furthermore, a yeast one-hybrid assay revealed that FaMYB5 influences citric acid accumulation by binding to the FaACO (aconitase), FaGAD (glutamate decarboxylase), and FaCS2 (citrate synthase) promoters. Dual-luciferase assays were used to demonstrate that FaMYB5 could activate FaCS2 expression and repress the transcription levels of FaACO and FaGAD. This study identified important roles of FaMYB5 in the regulation of citric acid metabolism and provided a potential target for improving strawberry fruit taste in horticultural crops.