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γ-Aminobutyric acid (GABA) is a widely distributed non-protein amino acid that serves as a crucial inhibitory neurotransmitter in the brain, regulating various physiological functions. As a result of its potential benefits, GABA has gained substantial interest in the functional food and pharmaceutical industries. The enzyme responsible for GABA production is glutamic acid decarboxylase (GAD), which catalyzes the irreversible decarboxylation of glutamate. Understanding the crystal structure and catalytic mechanism of GAD is pivotal in advancing our knowledge of GABA production. This article provides an overview of GAD's sources, structure, and catalytic mechanism, and explores strategies for enhancing GABA production through fermentation optimization, metabolic engineering, and genetic engineering. Furthermore, the effects of GABA on the physiological functions of animal organisms are also discussed. To meet the increasing demand for GABA, various strategies have been investigated to enhance its production, including optimizing fermentation conditions to facilitate GAD activity. Additionally, metabolic engineering techniques have been employed to increase the availability of glutamate as a precursor for GABA biosynthesis. By fine-tuning fermentation conditions and utilizing metabolic and genetic engineering techniques, it is possible to achieve higher yields of GABA, thus opening up new avenues for its application in functional foods and pharmaceuticals. Continuous research in this field holds immense promise for harnessing the potential of GABA in addressing various health-related challenges.

Gamma-aminobutyric acid is one of the major inhibitory neurotransmitters in the nervous system. Although gamma-aminobutyric acid is commonly synthesized by chemical methods, its microbial biosynthesis is regarded as one of the best production methods among the conventional techniques. This study aimed to optimize and model the production of gamma-aminobutyric acid from Lactobacillus plantarum subsp. plantarum IBRC (10,817) under the influence of heat and ultrasonic shock using the response surface methodology. Heat and ultrasonic shock were applied in the lag phase of bacterial growth. Heat shock variables included heat treatment, monosodium glutamate concentration, and incubation time. Also, ultrasonic shock variables were ultrasonic intensity, ultrasonic time, incubation time, and monosodium glutamate concentration. By applying the 30.9 h of incubation, 3.082 g/L of monosodium glutamate, and thermal shock of 49.958 °C for 30 min, the production of 295.04 mg/L of gamma amino butyric acid was predicted. As for ultrasonic shock, using 3.28 (g/L) monosodium glutamate, 70 h of bacterial incubation, 7.7 min ultrasound shock, and ultrasound frequency of 26.58 kHz, the highest amount of metabolite production was anticipated to be 215.19 mg/L. Overall, it was found that the actual values were consistent with the predicted values., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter of the central nervous system that impacts physical and mental health. Low GABA levels have been documented in several diseases, including multiple sclerosis and depression, and studies suggest that GABA could improve disease outcomes in those conditions. Probiotic bacteria naturally produce GABA and have been engineered to enhance its synthesis. Strains engineered thus far use inducible expression systems that require the addition of exogenous molecules, which complicates their development as therapeutics. This study aimed to overcome this challenge by engineering Lactococcus lactis with a constitutive GABA synthesis gene cassette. GABA synthesizing and transport genes (gadB and gadC) were cloned onto plasmids downstream of constitutive L. lactis promoters [P2, P5, shortened P8 (P8s)] of different strengths and transformed into L. lactis. Fold increase in gadCB expression conferred by these promoters (P2, P5, and P8s) was 322, 422, and 627, respectively, compared to the unmodified strain (P = 0.0325, P8s). GABA synthesis in the highest gadCB expressing strain, L. lactis-P8s-glutamic acid decarboxylase (GAD), was dependent on media supplementation with glutamic acid and significantly higher than the unmodified strain (P < 0.0001, 125 mM, 200 mM glutamic acid). Lactococcus lactis-P8s-GAD is poised for therapeutic testing in animal models of low-GABA-associated disease., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

γ-Aminobutyric acid (GABA) is a crucial neurotransmitter with wide application prospects. In this study, we focused on a GABA-producing strain from a traditional Chinese fermented beverage system. Among the six isolates, Lactobacillus hilgardii GZ2 exhibited the greatest ability to produce GABA in the traditional Chinese fermented beverage system. To increase GABA production, we optimized carbon sources, nitrogen sources, temperature, pH, and monosodium glutamate and glucose concentrations and conducted fed-batch fermentation. The best carbon and nitrogen sources for GABA production and cell growth were glucose, yeast extract and tryptone. Gradual increases in GABA were observed as the glucose and monosodium glutamate concentrations increased from 10 g/L to 50 g/L. During fed-batch fermentation, lactic acid was used to maintain the pH at 5.56, and after feeding with 0.03 g/mL glucose and 0.4 g/mL sodium glutamate for 72 h, the GABA yield reached 239 g/L. This novel high-GABA-producing strain holds great potential for the industrial production of GABA, as well as the development of health-promoting functional foods and medical fields., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Genome-scale metabolic model (GEM) can be used to simulate cellular metabolic phenotypes under various environmental or genetic conditions. This study utilized the GEM to observe the internal metabolic fluxes of recombinant Escherichia coli producing gamma-aminobutyric acid (GABA). Recombinant E. coli was cultivated in a fermenter under three conditions: pH 7, pH 5, and additional succinic acids. External fluxes were calculated from cultivation results, and internal fluxes were calculated through flux optimization. Based on the internal flux analysis, glycolysis and pentose phosphate pathways were repressed under cultivation at pH 5, even though glutamate dehydrogenase increased GABA production. Notably, this repression was halted by adding succinic acid. Furthermore, proper sucA repression is a promising target for developing strains more capable of producing GABA.

Gamma-aminobutyric acid (γ-ABA) can be produced by various microorganisms including bacteria, fungi and yeasts using enzymatic bioconversion, microbial fermentation or chemical hydrolysis. Regenerating conjugated glycerol-amines is valid by the intervention of microbial cyclooxygenase [COX] and lipooxygenase [LOX] enzymes produced via lactobacillus bacteria (LAB) as successor enzymes to glutamate decarboxylases (GAD). Therefore, the aim of this review is to provide an overview on γ-ABA production, and microbiological achievements used in producing this signal molecule based on those fermenting enzymes. The formation of aminoglycerides based conjugated γ-ABA is considered the key substances in controlling the host defense against pathogens and is aimed in increasing the neurotransmission effects and in suppressing further cardiovascular diseases., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Psychobiotics are a novel class of probiotics with potential to confer mental wellness via production of neuroactive compounds such as gamma-aminobutyric acid (GABA). The demand for new biological sources of GABA has increased steadily. Therefore, the current study reports the isolation of 17 presumptive lactic acid bacteria (LAB) from marine samples and their screening for GABA synthesis from monosodium glutamate (MSG) using thin-layer chromatography (TLC). The isolate SH9 was selected as a high GABA producing strain. The GABA content of SH9 cell free supernatant (CFS) was quantitatively determined by high performance liquid chromatography (HPLC) to be 0.97 g/L. SH9 was identified biochemically and molecularly as Enterococcus faecium (identity 99%). Moreover, SH9 demonstrated promising probiotic potentials; it gave no signs of hemolysis and could survive at low pH values and high bile salt concentrations. It also exhibited antimicrobial activity against highly pathogenic strains and the ability to grow at 6.5% NaCl. In addition, SH9 CFS showed anti-inflammatory and antioxidant properties. The glutamate decarboxylase (GAD) gene was detected in SH9 by using specific primers. Product of 540 bp was obtained, sequenced, and analyzed (accession number: MW713382). The inferred amino acid sequence was 99.3% identical to Lactobacillus plantarum M-6 gadB gene. The findings of this study suggest that the marine isolate E. faecium SH9 could be used as a novel psychobiotics in the development of GABA rich healthy products., (© 2022. The Author(s).)

Gamma-aminobutyric acid (GABA) is a non-proteinogenic amino acid act as a major neurotransmitter inhibitor in the nervous system of mammals. It also used as a precursor of bioplastics synthesis such as N-methylpyrolidone and polyamide 4. Chemical-based synthesis methods have many environmental-related issues, so efforts have been made to develop biosynthetic methods to produce GABA. Glutamate decarboxylase (GAD) transforms L-glutamate to GABA using pyridoxal 5'-phosphate (PLP) as a cofactor. Bioconversion of GABA with whole cells overexpressing the glutamate decarboxylase has advantages of fewer byproducts and rapid reaction. However, there is a bottleneck in the whole-cell bioconversion system i.e., higher GABA production require a large amount of cofactor PLP which make the process costly. Therefore, pyridoxal kinase (PdxY) able to regenerate PLP was introduced in the whole-cell system to construct a new GABA producing system. Culture and reaction conditions were optimized, and 100% conversion of 0.6 M MSG was obtained. This study reports that a competitive level of GABA production could be achieved without supplying additional PLPs., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Glutamate decarboxylase (GAD; EC 4.1.1.15) catalyzes the irreversible decarboxylation of glutamate to produce γ-aminobutyric acid (GABA); a ubiquitous non-protein amino acid involved in the regulation of several aspects of plant metabolism and physiology. To study the function of GAD and GABA in maize, we have; 1) introduced native and deregulated forms of AtGAD1 into maize with the intent of increasing the synthesis of GABA and 2) introduced constructs into maize designed to suppress the activity of several GABA shunt, GABA transport and GABA pathway genes. Maize plants expressing the deregulated AtGAD1 exhibit a severe chlorosis and retarded growth phenotype and have high levels of GABA, and Ca++/CaM-independent GAD activity. Plants expressing the suppression constructs for GABA biosynthetic and transport pathway genes had no observable phenotype whereas a knockout of GABA catabolic pathway genes led to growth and developmental defects under standard growth conditions. The implications of this study to our understanding of the action and function of GABA and GAD in crops are discussed., Competing Interests: The authors have declared that no competing interests exist.

Glutamate decarboxylase B (GadB) from Escherichia coli, an intrinsic pyridoxal 5'-phosphate (PLP)-dependent enzyme has been employed for 4-aminobutyric acid (GABA) biosynthesis, which involves the glutamate import and GABA export via a transporter located in the inner membrane as rate determined step of whole-cell (WC) biotransformation. Herein, GadB was cloned and overexpressed in E. coli under a constitutive promoter in a high copy number plasmid, and 46.9 g/L GABA was produced. It was observed that GadB migrated to the periplasm when the WC were subjected to -20 °C cold treatment for 24 h prior to the biotransformation. Kinetic studies indicated that the enzymatic turnover rate of WC increased 2-fold after cold treatment, which was correlated with the migration rate of GadB, and up to 88.6% of GadB. The export or possible migration of GadB mitigated the rate-limiting step of WC biotransformation, and a 100% conversion of substrate to GABA was obtained. Finally, we launched a promising strategy for GABA production of 850 g/L from cost-effective monosodium glutamate (MSG) by using WC biocatalysts with 10-times recycling., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The identity of the mechanism that controls aggressive behavior in rodents is unclear. Serotonin (5-HT) and GABA are associated with aggressive behavior in rodents. However, the regulatory relationship between these chemicals in the different brain regions of rats has not been fully defined. This study aimed to clarify the role of GABABR1 in DRN-mediated GABA to regulate 5-HT expression in multiple brain regions in male rats with high and low aggressive behavior. Rat models of highly and less aggressive behavior were established through social isolation plus resident intruder. On this basis, GABA content in the DRN and 5-HT contents in the PFC, hypothalamus, hippocampus and DRN were detected using ELISA. Co-expression of 5-HT and GB1 in the DRN was detected by immunofluorescence and immunoelectron microscopy at the tissue and subcellular levels, respectively. GB1-specific agonist baclofen and GB1-specific inhibitor CGP35348 were injected into the DRN by stereotaxic injection. Changes in 5-HT levels in the PFC, hypothalamus and hippocampus were detected afterward. After modeling, rats with highly aggressive behavior exhibited higher aggressive behavior scores, shorter latencies of aggression, and higher total distances in the open field test than rats with less aggressive behavior. The contents of 5-HT in the PFC, hypothalamus and hippocampus of rats with high and low aggressive behavior (no difference between the two groups) were significantly decreased, but the change in GABA content in the DRN was the opposite. GB1 granules could be found on synaptic membranes containing 5-HT granules, which indicated that 5-HT neurons in the DRN co-expressed with GB1, which also occurred in double immunofluorescence results. At the same time, we found that the expression of GB1 in the DRN of rats with high and low aggressive behavior was significantly increased, and the expression of GB1 in the DRN of rats with low aggressive behavior was significantly higher than that in rats with high aggressive behavior. Nevertheless, the expression of 5-HT in DRN was opposite in these two groups. After microinjection of baclofen into the DRN, the 5-HT contents in the PFC, hypothalamus and hippocampus of rats in each group decreased significantly. In contrast, the 5-HT contents in the PFC, hypothalamus and hippocampus of rats in each group increased significantly after injection with CGP35348. The significant increase in GABA in the DRN combined with the significant increase in GB1 in the DRN further mediated the synaptic inhibition effect, which reduced the 5-HT level of 5-HT neurons in the DRN, resulting in a significant decrease in 5-HT levels in the PFC, hypothalamus and hippocampus. Therefore, GB1-mediated GABA regulation of 5-HT levels in the PFC, hypothalamus and hippocampus is one of the mechanisms of highly and less aggressive behavior originating in the DRN. The increased GB1 level in the DRN of LA-behavior rats exhibited a greater degree of change than in the HA-group rats, which indicated that differently decreased 5-HT levels in the DRN may be the internal mechanisms of high and low aggression behaviors., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu 1-3 . Here we identify the metabolite and neurotransmitter GABA as a candidate signalling molecule synthesized and secreted by activated B cells and plasma cells. We show that B cell-derived GABA promotes monocyte differentiation into anti-inflammatory macrophages that secrete interleukin-10 and inhibit CD8 + T cell killer function. In mice, B cell deficiency or B cell-specific inactivation of the GABA-generating enzyme GAD67 enhances anti-tumour responses. Our study reveals that, in addition to cytokines and membrane proteins, small metabolites derived from B-lineage cells have immunoregulatory functions, which may be pharmaceutical targets allowing fine-tuning of immune responses., (© 2021. The Author(s).)

Gamma-aminobutyric acid (GABA) plays a key role as an inhibitory neurotransmitter in the mammalian sympathetic nervous system and has other health benefits. Molecular characterization, genome analysis, and optimization were investigated to improve GABA production of a selected strain of lactic acid bacteria. Eleven isolates from plant materials were screened for GABA productivity and were identified based on phenotypic and genotypic characteristics. The most potent strain was chosen for genome analysis and GABA production optimization using the response surface methodology (RSM). Each of the two strains was closely related to Lactobacillus plantarum, Lactobacillus brevis, Weissella cibaria, Leuconostoc pseudomesenteroides while each strain was similar to Lactobacillus pentosus, Enterococcus lactis, and Leuconostoc mesenteroides. They produced GABA ranging from 0.036 ± 0.000 to 17.315 ± 0.171 g/L at 72 h-cultivation. Among them, the most potent strain, SL9-6, showed the highest GABA production (17.315 g/L) when cultivated with 10% (v/v) inoculum for 48 h. The draft genome sequence of strain SL9-6 exhibited 96.90% average nucleotide identity value and 74.50% digital DNA-DNA hybridization to Lactobacillus brevis NCTC 13768 T . This strain contained a glutamate decarboxylase gene system (gadA, gadB, and gadC). Optimal culture conditions were determined as 40.00 g/L glucose, 49.90 g/L monosodium glutamate, pH 5.94, and 31.10°C by RSM, giving maximum GABA production of 32.48 g/L. Results from RSM also indicated that monosodium glutamate concentration, pH, and temperature were significant variables. GABA production significantly improved here could promise further application of strain SL9-6.

Lactic acid bacteria (Lactobacillus plantarum KCTC 3103) were fermented to produce gamma-aminobutyric acid (GABA). The conditions of the modified synthetic medium were optimized as 5 g/L glucose, 10 g/L yeast extract, 100 g/L rice bran extract, and 1.0 g/L ascorbic acid for GABA production. Single-step fermentation of cell growth and GABA production with a modified synthetic medium was higher than those with an MRS medium. Two-step fermentation was evaluated by separating the cell growth and GABA production under a modified synthetic medium. The cell concentration of 1.65 g dcw/L produced by the modified synthetic medium was higher than that of 1.0 g dcw/L produced by the MRS medium at 36 h from the first step of two-step fermentation. The highest GABA production of L. plantarum KCTC 3103 was 0.67 g/L with monosodium glutamate addition at 60 h in the second step of fermentation. Two-step fermentation with the modified synthetic medium is suitable for GABA production because of its high GABA productivity and favorable cell growth., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The medium-chain fatty acids octanoic acid (C8) and decanoic acid (C10) are gaining attention as beneficial brain fuels in several neurological disorders. The protective effects of C8 and C10 have been proposed to be driven by hepatic production of ketone bodies. However, plasma ketone levels correlates poorly with the cerebral effects of C8 and C10, suggesting that additional mechanism are in place. Here we investigated cellular C8 and C10 metabolism in the brain and explored how the protective effects of C8 and C10 may be linked to cellular metabolism. Using dynamic isotope labeling, with [U- 13 C]C8 and [U- 13 C]C10 as metabolic substrates, we show that both C8 and C10 are oxidatively metabolized in mouse brain slices. The 13 C enrichment from metabolism of [U- 13 C]C8 and [U- 13 C]C10 was particularly prominent in glutamine, suggesting that C8 and C10 metabolism primarily occurs in astrocytes. This finding was corroborated in cultured astrocytes in which C8 increased the respiration linked to ATP production, whereas C10 elevated the mitochondrial proton leak. When C8 and C10 were provided together as metabolic substrates in brain slices, metabolism of C10 was predominant over that of C8. Furthermore, metabolism of both [U- 13 C]C8 and [U- 13 C]C10 was unaffected by etomoxir indicating that it is independent of carnitine palmitoyltransferase I (CPT-1). Finally, we show that inhibition of glutamine synthesis selectively reduced 13 C accumulation in GABA from [U- 13 C]C8 and [U- 13 C]C10 metabolism in brain slices, demonstrating that the glutamine generated from astrocyte C8 and C10 metabolism is utilized for neuronal GABA synthesis. Collectively, the results show that cerebral C8 and C10 metabolism is linked to the metabolic coupling of neurons and astrocytes, which may serve as a protective metabolic mechanism of C8 and C10 supplementation in neurological disorders., (© 2021. The Author(s).)

The second messenger 2'3'-cyclic-GMP-AMP (cGAMP) is thought to be transmitted from brain carcinomas to astrocytes via gap junctions, which functions to promote metastasis in the brain parenchyma. In the current study, we established a method to introduce cGAMP into astrocytes, which simulates the state of astrocytes that have been invaded by cGAMP around tumors. Astrocytes incorporating cGAMP were analyzed by metabolomics, which demonstrated that cGAMP increased glutamate production and astrocyte secretion. The same trend was observed for γ-aminobutyric acid (GABA). Conversely, glutamine production and secretion were decreased by cGAMP treatment. Due to the fundamental role of astrocytes in regulation of the glutamine-glutamate cycle, such metabolic changes may represent a potential mechanism and therapeutic target for alteration of the central nervous system (CNS) environment and the malignant transformation of brain carcinomas.

Background: Gamma-aminobutyric acid (GABA) is an important bio-product used in pharmaceuticals and functional foods and as a precursor of the biodegradable plastic polyamide 4. Glutamate decarboxylase (GAD) converts L-glutamate (L-Glu) into GABA via decarboxylation. Compared with other methods, develop a bioconversion platform to produce GABA is of considerable interest for industrial use., Results: Three GAD genes were identified from three Bacillus strains and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction temperature and pH values for three enzymes were 40 °C and 5.0, respectively. Of the GADs, GADZ11 had the highest catalytic efficiency towards L-Glu (2.19 mM - 1  s - 1 ). The engineered E. coli strain that expressed GADZ11 was used as a whole-cell biocatalyst for the production of GABA. After repeated use 14 times, the cells produced GABA with an average molar conversion rate of 98.6% within 14 h., Conclusions: Three recombinant GADs from Bacillus strains have been conducted functional identification. The engineered E. coli strain heterologous expressing GADZ1, GADZ11, and GADZ20 could accomplish the biosynthesis of L-Glu to GABA in a buffer-free reaction at a high L-Glu concentration. The novel engineered E. coli strain has the potential to be a cost-effective biotransformation platform for the industrial production of GABA., (© 2021. The Author(s).)

Purpose: Polycystic ovary syndrome (PCOS) is a common heterogeneous endocrine disorder companied with neuroendocrine and metabolic disorders. Gut microbiota has been implicated to play a key role in metabolic diseases and the production of neurotransmitters. Previous studies have reported the alterations in the gut microbiota of PCOS patients and animal models, however, most of the articles did not take the effect of age or diet on gut microbiota into account. The aim of this study was to identify the differential gut microbial species in PCOS patients compared with age and BMI-matched healthy control women., Methods: We performed physical examinations and dietary survey in 20 women with PCOS (lean PCOS, PL, n = 10; overweight PCOS, PO, n = 10) and 20 healthy control women (lean control, CL, n = 10; overweight control, CO, n = 10), and collected the blood on the days 1-3 of the menstrual cycle for the measurement of endocrine and metabolic profiles, and inflammatory factors; and collected the feces in non-menstrual period to investigate the composition of gut microbiota by sequencing the V4 region of the 16S rDNA gene in fecal samples. The correlations between clinical parameters and the differential species were evaluated., Results: Dietary analysis showed that the intake of dietary fiber, vitamin D were significantly decreased in PCOS. For the first time, our study found an increase of gamma-aminobutyric acid (GABA)-producing species in PCOS, including Parabacteroides distasonis, Bacteroides fragilis and Escherichia coli, which significantly positively correlated with serum LH levels and LH:FSH ratios., Conclusions: GABA-producing bacteria that were increased in PCOS, including Parabacteroides distasonis, Bacteroides fragilis and Escherichia coli, showed positive relationship with serum LH levels and LH:FSH ratios. In conclusion, gut microbial dysbiosis in women with PCOS is associated with neuroendocrine changes, revealing a potential gut-brain axis in PCOS., (© 2021. Italian Society of Endocrinology (SIE).)

Gamma-aminobutyric acid (GABA) is a principal inhibitory neurotransmitter in the central nervous system and is produced by irreversible decarboxylation of glutamate. It possesses several physiological functions such as neurotransmission, diuretic, and tranquilizer effects and also regulates cardiovascular functions such as blood pressure and heart rate in addition to playing a role in the reduction of pain and anxiety. The objective of this study was to evaluate the GABA producing ability and probiotic capability of certain lactic acid bacteria strains isolated from dairy products. Around sixty-four bacterial isolates were collected and screened for their ability to produce GABA from monosodium glutamate, among which nine isolates were able to produce GABA. The most efficient GABA producer was Enterococcus faecium BS5. Further, assessment of several important and desirable probiotic properties showed that Ent. faecium BS5 was resistant to acid stress, bile salt, and antibiotics. Ent. faecium BS5 may potentially be used for large-scale industrial production of GABA and also for functional fermented product development., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The compound γ-aminobutyric acid (GABA) was widely used in various fields. To enhance the production of GABA in Escherichia coli BL21(DE3), the enzymes of the regeneration pathway of the coenzyme factor pyridoxal 5'-phosphate (PLP) were engineered. The recombinant E. coli strain was screened and identified. The initial concentrations of L-monosodium glutamate (L-MSG) had an obvious influence on the production of GABA. The highest concentration of GABA in recombinant E. coli BL21/pET28a-gadA was 5.54 g/L when the initial L-MSG concentration was 10 g/L, whereas it was 8.45 g/L in recombinant E. coli BL21/pET28a-gadA-SNO1-SNZ1 at an initial L-MSG concentration of 15 g/L. The corresponding conversion yields of GABA in these two strains were 91.0% and 92.7%, respectively. When the initial concentrations of L-MSG were more than 15 g/L, the concentrations of GABA in E. coli BL21/pET28a-gadA-SNO1-SNZ1 were significantly higher as compared to those in recombinant E. coli BL21/pET28a-gadA, and it reached a maximum of 13.20 g/L at an initial L-MSG concentration of 25 g/L, demonstrating that the introduction of the enzymes of the regeneration pathway of PLP favored to enhance the production of GABA. This study provides new insight into producing GABA effectively in E. coli BL21(DE3).

Monoamine oxidase (MAO) is believed to mediate the degradation of monoamine neurotransmitters, including dopamine, in the brain. Between the two types of MAO, MAO-B has been believed to be involved in dopamine degradation, which supports the idea that the therapeutic efficacy of MAO-B inhibitors in Parkinson's disease can be attributed to an increase in extracellular dopamine concentration. However, this belief has been controversial. Here, by utilizing in vivo phasic and basal electrochemical monitoring of extracellular dopamine with fast-scan cyclic voltammetry and multiple-cyclic square wave voltammetry and ex vivo fluorescence imaging of dopamine with GRAB DA2m , we demonstrate that MAO-A, but not MAO-B, mainly contributes to striatal dopamine degradation. In contrast, our whole-cell patch-clamp results demonstrated that MAO-B, but not MAO-A, was responsible for astrocytic GABA-mediated tonic inhibitory currents in the rat striatum. We conclude that, in contrast to the traditional belief, MAO-A and MAO-B have profoundly different roles: MAO-A regulates dopamine levels, whereas MAO-B controls tonic GABA levels., (© 2021. The Author(s).)

When synchronized with the light/dark cycle the circadian rhythm is termed a diurnal rhythm and this organizes an organism's daily life cycle in relation to the metabolic shifts during the day/night cycles. This is a complex task, particularly under stress conditions. Accurate maintenance of the diurnal rhythm becomes an issue under environmental extremes, such as drought due to the impairment of metabolism, redox balance, and structural integrity. In plants, the non-proteinogenic amino acid GABA accumulates to high levels in response to several stress factors but this is not always dependent on the activation of its biosynthesis. Here we propose a regulatory role to GABA during the diurnal rhythm in plants which is similar to its function in animals where it adjusts the circadian rhythm. Here we investigated whether GABA-biosynthesis was affected by drought stress during the diurnal cycle. For this, we took samples from leaves of N. tabacum plants subjected to PEG-mediated drought stress (-0.73 MPa) during the day and night cycle during a 24 hour period. Glutamate, GABA, and proline contents, along with GDH, GAD enzyme activities and transcript profiles were analyzed. Overall, we conclude that the oscillations in GABA biosynthesis during day and night cycle have an impact on drought stress responses which needs to be elucidated by further analysis.

This study aimed to enhance natural gamma aminobutyric acid (GABA) production in yoghurt by the addition of simple sugars and commercial prebiotics without the need for pyridoxal 5'-phosphate (PLP) cofactor. The simple sugars induced more GABA production (42.83-58.56 mg/100 g) compared to the prebiotics (34.19-40.51 mg/100 g), with glucose promoting the most GABA production in yoghurt (58.56 mg/100 g) surpassing the control sample with added PLP (48.01 mg/100 g). The yoghurt prepared with glucose also had the highest probiotic count (9.31 log CFU/g). Simulated gastrointestinal digestion of this GABA-rich yoghurt showed a non-significant reduction in GABA content and probiotic viability, demonstrating the resistance towards a highly acidic environment (pH 1.2). Refrigerated storage up to 28 days improved GABA production (83.65 mg/100 g) compared to fresh GABA-rich yoghurt prepared on day 1. In conclusion, the addition of glucose successfully mitigates the over-use of glutamate and omits the use of PLP for increased production of GABA in yoghurt, offering an economical approach to produce a probiotic-rich dairy food with potential anti-hypertensive effects.

Mycorrhizas are known to have a positive impact on plant growth and ability to resist major biotic and abiotic stresses. However, the metabolic alterations underlying mycorrhizal symbiosis are still understudied. By using metabolomics and transcriptomics approaches, cork oak roots colonized by the ectomycorrhizal fungus Pisolithus tinctorius were compared with non-colonized roots. Results show that compounds putatively corresponding to carbohydrates, organic acids, tannins, long-chain fatty acids and monoacylglycerols, were depleted in ectomycorrhizal cork oak colonized roots. Conversely, non-proteogenic amino acids, such as gamma-aminobutyric acid (GABA), and several putative defense-related compounds, including oxylipin-family compounds, terpenoids and B6 vitamers were induced in mycorrhizal roots. Transcriptomic analysis suggests the involvement of GABA in ectomycorrhizal symbiosis through increased synthesis and inhibition of degradation in mycorrhizal roots. Results from this global metabolomics analysis suggest decreases in root metabolites which are common components of exudates, and in compounds related to root external protective layers which could facilitate plant-fungal contact and enhance symbiosis. Root metabolic pathways involved in defense against stress were induced in ectomycorrhizal roots that could be involved in a plant mechanism to avoid uncontrolled growth of the fungal symbiont in the root apoplast. Several of the identified symbiosis-specific metabolites, such as GABA, may help to understand how ectomycorrhizal fungi such as P. tinctorius benefit their host plants.

Visuospatial working memory (vsWM) requires information transfer among multiple cortical regions, from primary visual (V1) to prefrontal (PFC) cortices. This information is conveyed via layer 3 glutamatergic neurons whose activity is regulated by gamma-aminobutyric acid (GABA)ergic interneurons. In layer 3 of adult human neocortex, molecular markers of glutamate neurotransmission were lowest in V1 and highest in PFC, whereas GABA markers had the reverse pattern. Here, we asked if these opposite V1-visual association cortex (V2)-posterior parietal cortex (PPC)-PFC gradients across the vsWM network are present in layer 3 of monkey neocortex, when they are established during postnatal development, and if they are specific to this layer. We quantified transcript levels of glutamate and GABA markers in layers 3 and 6 of four vsWM cortical regions in a postnatal developmental series of 30 macaque monkeys. In adult monkeys, glutamate transcript levels in layer 3 increased across V1-V2-PPC-PFC regions, whereas GABA transcripts showed the opposite V1-V2-PPC-PFC gradient. Glutamate transcripts established adult-like expression patterns earlier during postnatal development than GABA transcripts. These V1-V2-PPC-PFC gradients and developmental patterns were less evident in layer 6. These findings demonstrate that expression of glutamate and GABA transcripts differs across cortical regions and layers during postnatal development, revealing potential molecular substrates for vsWM functional maturation., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

The antimalarial artemisinins have also been implicated in the regulation of various cellular pathways including immunomodulation of cancers and regulation of pancreatic cell signaling in mammals. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the postsynaptic scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin B6 (also known as pyridoxal 5'-phosphate [PLP]), in complex with artesunate at 2.4-Å resolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins also interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain., Competing Interests: The authors declare no competing interest.

Glutamate decarboxylase (GAD, EC 4.1.1.15) is an important enzyme in gamma-aminobutyric acid biosynthesis and DL-glutamic acid resolution. In this study, the Enterococcus faecium-derived GAD was successfully immobilized by regenerated chitin (RC) via specific adsorption of cellulose-binding domain (CBD). The optimal binding buffer was 20 mmol/L phosphate buffer saline (pH 8.0), and the RC binding capacity was 1.77 ± 0.11 mg cbd-gad /g rc under this condition. The ratio of wet RC and crude enzyme solution used for immobilization was recommended to 3:50 (g/mL). To evaluate the effect of RC immobilization on GAD, properties of the immobilize GAD (RC-CBD-GAD) were investigated. Results indicated RC-CBD-GAD was relatively stable at pH 4.4-5.6 and temperature - 20-40 °C, and the optimal reaction pH value and temperature were pH 4.8 and 50 °C, respectively. When it was reacted with 5 mmol/L of follow chemical reagents respectively, the activity of RC-CBD-GAD was hardly affected by EDTA, KCl, and NaCl, and significantly inactivated by AgNO 3 , MnSO 4 , MgSO 4 , CuSO 4 , ZnSO 4 , FeCl 2 , FeCl 3 , AlCl 3 , CaCl 2, and Pb(CH 3 COO) 2 . The apparent K m and V max were 28.35 mmol/L and 147.06 μmol/(g RC-CBD-GAD ·min), respectively. The optimum time for a batch of catalytic reaction without exogenous pH control was 2 h. Under this reaction time, RC-CBD-GAD had a good reusability with a half-life of 23 cycles, indicating that it was very attractive for GABA industry. As a novel, efficient, and green CBD binding carrier, RC provides an alternative way to protein immobilization.

Sensory discrimination is essential for survival. However, how sensory information is finely controlled in the brain is not well defined. Here, we show that astrocytes control tactile acuity via tonic inhibition in the thalamus. Mechanistically, diamine oxidase (DAO) and the subsequent aldehyde dehydrogenase 1a1 (Aldh1a1) convert putrescine into GABA, which is released via Best1. The GABA from astrocytes inhibits synaptically evoked firing at the lemniscal synapses to fine-tune the dynamic range of the stimulation-response relationship, the precision of spike timing, and tactile discrimination. Our findings reveal a novel role of astrocytes in the control of sensory acuity through tonic GABA release., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Gamma aminobutyric acid (GABA) is a non-protein amino acid that is widely distributed in nature and its physiological importance goes beyond its role as an inhibitory neurotransmitter of the central nervous system in mammals. Since microbial fermentation is one of the most promising methods to obtain GABA, the production of this metabolite by several strains of lactic acid bacteria isolated from quinoa and amaranth sourdoughs was investigated. Lactobacillus brevis CRL 2013 produced the highest GABA levels, reaching 265 mM when optimal culture conditions were set up. The fermentative profile showed that CRL 2013 was able to catabolize carbohydrates through the phosphoketolase pathway yielding variable amounts of lactic acid, acetate and ethanol, which depended on the type of carbon source available and the presence of external electron acceptors such as fructose. Enhanced growth parameters and low GABA synthesis were associated to pentose fermentation. This impairment on GABA production machinery was partially overpassed by the addition of ethanol to the culture media. These results support the potential use of L. brevis CRL 2013 as a starter culture for the manufacture of GABA-enriched functional foods and provide further insights to the understanding of the GAD system regulation in lactic acid bacteria., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Tonic spinal cord stimulation (SCS) has been used as a treatment for chronic neuropathic pain ever since its discovery in late 1960s. Despite its clinical successes in a subset of chronic neuropathic pain syndromes, several limitations such as insufficient pain relief and uncomfortable paresthesias have led to the development of new targets, the dorsal root ganglion, and new stimulation waveforms, such as burst and high frequency. The aim of this review is to provide a brief overview of the main mechanisms behind the mode of action of the different SCS paradigms. Tonic SCS mainly acts via a segmental spinal mechanism where it induces GABA-release from inhibitory interneurons in the spinal dorsal horn. Tonic SCS concurrently initiates neuropathic pain modulation through a supraspinal-spinal feedback loop and serotonergic descending fibers. Mechanisms of stimulation of the DRG as well as those related to new SCS paradigms are now under investigation, where it seems that burst SCS not only stimulates sensory, discriminative aspects of pain (like Tonic SCS) but also emotional, affective, and motivational aspects of pain. Initial long-term study results on closed-loop SCS systems hold promise for improvement of future SCS treatment.

Today, a number of studies conclusively show that certain bacterial strains, mainly from the genera Lactobacillus and Bifidobacterium, influence the functioning of the central nervous system, leading to changes in beahvior, nociception and the cognitive abilities of humans and animals. Such strains serve as the basis for developing probiotics with a curative potential for the central nervous system - psychobioitcs. However, the question of how to find such strains and which criteria to use for their selection remains unanswered. Some compounds produced by bacteria, such as gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter of the central nervous system, are potential mediators between bacterial cells and the host. Previously, we established that some species of Lactobacillus and Bifidobacterium are capable of producing GABA. We presumed that GABA-producing Lactobacillus and Bifidobacterium strains are great candidates to use as psychobiotics. Therefore, we selected the strains Lactobacillus plantarum 90sk and Bifidobacterium adolescentis 150 as efficient GABA producers. The goal of this work was to assess the probiotic properties of the selected strains as well as their antidepressive effects in mice. We established that the ingestion of the probiotic composition based on the selected strains by BALB/c mice for 2 weeks reduced depressive-like behavior in the forced swimming test; the effect was similar to that of fluoxetine.

Gamma aminobutyric acid (GABA) is the principal inhibitory neurotransmitter playing a key role in anxiety and depression disorders in mammals. Recent studies revealed that members of the gut microbiota are able to produce GABA modulating the gut-brain axis response. Among members of the human gut microbiota, bifidobacteria are well known to establish many metabolic and physiologic interactions with the host. In this study, we performed genome analyses of more than 1,000 bifidobacterial strains publicly available revealing that Bifidobacterium adolescentis taxon might represent a model GABA producer in human gastrointestinal tract. Moreover, the in silico screening of human/animal metagenomic datasets showed an intriguing association/correlation between B. adolescentis load and mental disorders such as depression and anxiety. Interestingly, in vitro screening of 82 B. adolescentis strains allowed identifying two high GABA producers, i.e. B. adolescentis PRL2019 and B. adolescentis HD17T2H, which were employed in an in vivo trial in rats. Feeding Groningen rats with a supplementation of B. adolescentis strains, confirmed the ability of these microorganisms to stimulate the in vivo production of GABA highlighting their potential implication in gut-brain axis interactions.

GABA signaling is involved in a wide range of neuronal functions, such as synchronization of action potential firing, synaptic plasticity and neuronal development. Sustained GABA signaling requires efficient mechanisms for the replenishment of the neurotransmitter pool of GABA. The prevailing theory is that exocytotically released GABA may be transported into perisynaptic astroglia and converted to glutamine, which is then shuttled back to the neurons for resynthesis of GABA-i.e., the glutamate/GABA-glutamine (GGG) cycle. However, an unequivocal demonstration of astroglia-to-nerve terminal transport of glutamine and the contribution of astroglia-derived glutamine to neurotransmitter GABA synthesis is lacking. By genetic inactivation of the amino acid transporter Solute carrier 38 member a1 (Slc38a1)-which is enriched on parvalbumin + GABAergic neurons-and by intraperitoneal injection of radiolabeled acetate (which is metabolized to glutamine in astroglial cells), we show that Slc38a1 mediates import of astroglia-derived glutamine into GABAergic neurons for synthesis of GABA. In brain slices, we demonstrate the role of Slc38a1 for the uptake of glutamine specifically into GABAergic nerve terminals for the synthesis of GABA depending on demand and glutamine supply. Thus, while leaving room for other pathways, our study demonstrates a key role of Slc38a1 for newly formed GABA, in harmony with the existence of a GGG cycle.

Gamma-Aminobutyric acid (GABA) is a non-protein amino acid widely distributed in nature. It is produced through irreversible α-decarboxylation of glutamate by enzyme glutamate decarboxylase (GAD). GABA and GAD have been found in plants, animals, and microorganisms. GABA is distributed throughout the human body and it is involved in the regulation of cardiovascular conditions such as blood pressure and heart rate, and plays a role in the reduction of anxiety and pain. Although researchers had produced GABA by chemical method earlier it became less acceptable as it pollutes the environment. Researchers now use a more promising microbial method for the production of GABA. In the drug and food industry, demand for GABA is immense. So, large scale conversion of GABA by microbes has got much attention. So this review focuses on the isolation source, production, and functions of GABA in the microbial system. We also summarize the mechanism of action of GABA and its shunt pathway.

Background: Gamma aminobutyric acid (GABA) is an important platform chemical, which has been used as a food additive and drug. Additionally, GABA is a precursor of 2-pyrrolidone, which is used in nylon synthesis. GABA is usually synthesized from glutamate in a reaction catalyzed by glutamate decarboxylase (GAD). Currently, there are several reports on GABA production from monosodium glutamate (MSG) or glucose using engineered microbes. However, the optimal pH for GAD activity is 4, which is the limiting factor for the efficient microbial fermentative production of GABA as fermentations are performed at pH 7. Recently, DR1558, a response regulator in the two-component signal transduction system was identified in Deinococcus radiodurans. DR1558 is reported to confer cellular robustness to cells by binding the promoter regions of genes via DNA-binding domains or by binding to the effector molecules, which enable the microorganisms to survive in various environmental stress conditions, such as oxidative stress, high osmotic shock, and low pH., Results: In this study, the effect of DR1558 in enhancing GABA production was examined using two different strategies: whole-cell bioconversion of GABA from MSG and direct fermentative production of GABA from glucose under acidic culture conditions. In the whole-cell bioconversion, GABA produced by E. coli expressing GadBC and DR1558 (6.52 g/L GABA from 13 g/L MSG·H 2 O) in shake flask culture at pH 4.5 was 2.2-fold higher than that by E. coli expressing only GadBC (2.97 g/L of GABA from 13 g/L MSG·H 2 O). In direct fermentative production of GABA from glucose, E. coli ∆gabT expressing isocitrate dehydrogenase (IcdA), glutamate dehydrogenase (GdhA), GadBC, and DR1558 produced 1.7-fold higher GABA (2.8 g/L of GABA from 30 g/L glucose) than E. coli ∆gabT expressing IcdA, GdhA, and GadBC (1.6 g/L of GABA from 30 g/L glucose) in shake flask culture at an initial pH 7.0. The transcriptional analysis of E. coli revealed that DR1558 conferred acid resistance to E. coli during GABA production. The fed-batch fermentation of E. coli expressing IcdA, GdhA, GadBC, and DR1558 performed at pH 5.0 resulted in the final GABA titer of 6.16 g/L by consuming 116.82 g/L of glucose in 38 h., Conclusion: This is the first report to demonstrate GABA production by acidic fermentation and to provide an engineering strategy for conferring acid resistance to the recombinant E. coli for GABA production.

Gamma-aminobutyric acid (GABA) is an industrially valuable natural product. This study was aimed to establish an efficient food-grade production process of GABA by engineering Saccharomyces cerevisiae that is generally recognized as safe (GRAS). GABA can be produced by catalytic decarboxylation of l-glutamate (l-Glu) by glutamate decarboxylase (GAD, EC4.1.1.15). Two GADs, SsGAD from Streptomyces sp. MJ654-NF4 and ScGAD from Streptomyces chromofuscus ATCC 49982, were heterologously expressed in S. cerevisiae BJ5464. The engineered yeast strains were used as whole-cell biocatalysts for GABA production. S. cerevisiae BJ5464/SsGAD exhibited significantly higher efficient catalytic activity than that of S. cerevisiae BJ5464/ScGAD. The optimal bioconversion system consisted of a cell density of OD 600 30, 0.1 M l-Glu, and 0.28 mM pyridoxal phosphate in 0.2 M Na 2 HPO 4 -citric acid buffer with pH 5.4, and the reactions were performed at 50 °C for 12 H. S. cerevisiae BJ5464/SsGAD cells can be reused, and the accumulated GABA titer reached 62.6 g/L after 10 batches with an overall molar conversion rate of 60.8 mol%. This work thus provides an effective production process of GABA using engineered yeast for food and pharmaceutical applications., (© 2019 International Union of Biochemistry and Molecular Biology, Inc.)

Current pharmacological treatments for Parkinson's disease (PD) are focused on symptomatic relief, but not on disease modification, based on the strong belief that PD is caused by irreversible dopaminergic neuronal death. Thus, the concept of the presence of dormant dopaminergic neurons and its possibility as the disease-modifying therapeutic target against PD have not been explored. Here we show that optogenetic activation of substantia nigra pars compacta (SNpc) neurons alleviates parkinsonism in acute PD animal models by recovering tyrosine hydroxylase (TH) from the TH-negative dormant dopaminergic neurons, some of which still express DOPA decarboxylase (DDC). The TH loss depends on reduced dopaminergic neuronal firing under aberrant tonic inhibition, which is attributed to excessive astrocytic GABA. Blocking the astrocytic GABA synthesis recapitulates the therapeutic effect of optogenetic activation. Consistently, SNpc of postmortem PD patients shows a significant population of TH-negative/DDC-positive dormant neurons surrounded by numerous GABA-positive astrocytes. We propose that disinhibiting dormant dopaminergic neurons by blocking excessive astrocytic GABA could be an effective therapeutic strategy against PD., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

A pathway for polyamine-derived GABA synthesis in Synechocystis sp. PCC 6803 was explored by disrupting both the glutamate decarboxylase and α-ketoglutarate decarboxylase genes. The generated Δgad:Δkgd strain had increased intracellular α-ketoglutarate and polyamine levels compared to the wild type. Gene transcript analysis using RT-PCR indicated that the Δgad:Δkgd strain had up-regulated expression of a putative gadbh whose gene product, γ-aminobutanal dehydrogenase, would catalyze the conversion of γ-aminobutanal to GABA. A strain with disrupted gabdh showed an increase in GABA, glutamate, succinate and spermidine levels. These findings provide evidence for a link between spermidine degradation and GABA synthesis in cyanobacteria. This study highlights the role of γ-aminobutanal dehydrogenase in maintaining an intact tricarboxylic acid cycle in Synechocystis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pyridoxal-5'-phosphate, the active form of vitamin B 6 , is associated with activities of several enzymes and the treatment of various neurological disorders. Here, we investigated the effects of pyridoxine on the immunoreactivity and protein levels of γ-aminobutyric acid (GABA)-synthesizing and degradation enzymes such as glutamic acid decarboxylase (GAD), GABA transaminase (GABA-T), and succinic semialdehyde dehydrogenase (SSADH), in the hippocampus of mice. The mice intraperitonially received physiological saline and 350 mg/kg pyridoxine, twice a day for 21 days, and were euthanized 2 h after the final dose. In the vehicle-treated group, we observed GAD67 immunoreactivity in the stratum pyramidale of the CA1 and CA3 region, Schaffer collateral, polymorphic layer, and outer granule cell layer of the dentate gyrus. Pyridoxine administration significantly increased GAD67 immunoreactivity, while significantly decreasing GABA-T immunoreactivity in pyridoxine-treated mouse hippocampi (CA1 region and dentate gyrus). In the stratum lacunosum-moleculare of CA1 region, GABA-T immunoreactivity was significantly increased in the pyridoxine-treated group compared to that in the vehicle-treated group, although GAD67 immunoreactivity was similarly observed in these groups. Alternatively, there were no significant differences in SSADH immunoreactivity in any regions of the hippocampus between the vehicle- and pyridoxine-treated groups. Western blot analysis showed significant increases in GAD67 and GABA-T protein levels in the pyridoxine-treated group compared with those in the vehicle-treated group. Therefore, pyridoxine administration facilitates GABA turnover in mouse hippocampus by modulating the GABA-synthesizing and degradation enzymes., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

γ-Aminobutyric acid (GABA) neurons in the ventral tegmental area (VTA) provide local inhibitory control of dopamine neuron activity and send long-range projections to several target regions including the nucleus accumbens. They play diverse roles in reward and aversion, suggesting that they be comprised of several functionally distinct sub-groups, but our understanding of this diversity has been limited by a lack of molecular markers that might provide genetic entry points for cell type-specific investigations. To address this, we conducted transcriptional profiling of GABA neurons and dopamine neurons using immunoprecipitation of tagged polyribosomes (RiboTag) and RNAseq. First, we directly compared these two transcriptomes in order to obtain a list of genes enriched in GABA neurons compared with dopamine neurons. Next, we created a novel bioinformatic approach, that used the PANTHER (Protein ANalysis THrough Evolutionary Relationships) gene ontology database and VTA gene expression data from the Allen Mouse Brain Atlas, from which we obtained 6 candidate genes: Cbln4, Rxfp3, Rora, Gpr101, Trh and Nrp2. As a final step, we verified the selective expression of these candidate genes in sub-groups of GABA neurons in the VTA (and neighbouring substantia nigra pars compacta) using immunolabelling. Taken together, our study provides a valuable toolbox for the future investigation of GABA neuron sub-groups in the VTA., (© 2019 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Screening for various types of lactic acid bacteria (LAB) that form the biological agent γ-amino-butyric acid (GABA) is important to produce different kinds of GABA-containing fermented foods. So far, no GABA-producing LAB have been reported from Cambodian fermented foods. Most small-scale fermentations and even some industrial processes in this country still rely on indigenous LAB. The application of GABA-producing autochthonous starters would allow the production of Cambodian fermented foods with an additional nutritional value that meet the population's dietary habits and that are also more attractive for the international food market. Matrix-assisted laser desorption/ionizing time-of-flight mass spectrometry (MALDI-TOF MS) and partial 16S rDNA sequencing were used to identify 68 LAB isolates from Cambodian fermented foods. These isolates were classified and grouped with (GTG) 5 rep-PCR, resulting in 50 strains. Subsequently, all strains were investigated for their ability to produce GABA by thin layer chromatography. GABA-positive strains were further analyzed by the GABase assay. Of the six GABA-positive LAB strains-one Lactobacillus futsaii , two Lactobacillus namurensis , and three Lactobacillus plantarum strains-two Lactobacillus plantarum strains produced high amounts of GABA (20.34 mM, 16.47 mM). These strains should be further investigated for their potential application as GABA-producing starter cultures in the food applications.

Metabolic and neuroactive metabolite production represents one of the mechanisms through which the gut microbiota can impact health. One such metabolite, gamma-aminobutyric acid (GABA), can modulate glucose homeostasis and alter behavioural patterns in the host. We previously demonstrated that oral administration of GABA-producing Lactobacillus brevis DPC6108 has the potential to increase levels of circulating insulin in healthy rats. Therefore, the objective of this study was to assess the efficacy of endogenous microbial GABA production in improving metabolic and behavioural outcomes in a mouse model of metabolic dysfunction. Diet-induced obese and metabolically dysfunctional mice received one of two GABA-producing strains, L. brevis DPC6108 or L. brevis DSM32386, daily for 12 weeks. After 8 and 10 weeks of intervention, the behavioural and metabolic profiles of the mice were respectively assessed. Intervention with both L. brevis strains attenuated several abnormalities associated with metabolic dysfunction, causing a reduction in the accumulation of mesenteric adipose tissue, increased insulin secretion following glucose challenge, improved plasma cholesterol clearance and reduced despair-like behaviour and basal corticosterone production during the forced swim test. Taken together, this exploratory dataset indicates that intervention with GABA-producing lactobacilli has the potential to improve metabolic and depressive- like behavioural abnormalities associated with metabolic syndrome in mice.

Neural activation patterns in the ventral visual cortex in response to different categories of visual stimuli (e.g., faces vs. houses) are less selective, or distinctive, in older adults than in younger adults, a phenomenon known as age-related neural dedifferentiation. In this study, we investigated whether neural dedifferentiation extends to the auditory cortex. Inspired by previous animal work, we also investigated whether individual differences in GABA are associated with individual differences in neural distinctiveness in humans. 20 healthy young adults (ages 18-29) and 23 healthy older adults (over 65) completed a functional magnetic resonance imaging (fMRI) scan, during which neural activity was estimated while they listened to music and foreign speech. GABA levels in the auditory, ventrovisual and sensorimotor cortex were estimated in the same individuals in a separate magnetic resonance spectroscopy (MRS) scan. Relative to the younger adults, the older adults exhibited both (1) less distinct activation patterns for music vs. speech stimuli and (2) lower GABA levels in the auditory cortex. Also, individual differences in auditory GABA levels (but not ventrovisual or sensorimotor GABA levels) were associated with individual differences in neural distinctiveness in the auditory cortex in the older adults. These results demonstrate that age-related neural dedifferentiation extends to the auditory cortex and suggest that declining GABA levels may play a role in neural dedifferentiation in older adults., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Most microorganisms have evolved to maximize growth rate, with rapid consumption of carbon sources from the surroundings. However, fast growing phenotypes usually feature secretion of organic compounds. For example, E. coli mainly produced acetate in fast growing condition such as glucose rich and aerobic condition, which is troublesome for metabolic engineering because acetate causes acidification of surroundings, growth inhibition and decline of production yield. The overflow metabolism can be alleviated by reducing glucose uptake rate., Results: As glucose transporters or their subunits were knocked out in E. coli, the growth and glucose uptake rates decreased and biomass yield was improved. Alteration of intracellular metabolism caused by the mutations was investigated with transcriptome analysis and 13 C metabolic flux analysis ( 13 C MFA). Various transcriptional and metabolic perturbations were identified in the sugar transporter mutants. Transcription of genes related to glycolysis, chemotaxis, and flagella synthesis was downregulated, and that of gluconeogenesis, Krebs cycle, alternative transporters, quorum sensing, and stress induced proteins was upregulated in the sugar transporter mutants. The specific production yields of value-added compounds (enhanced green fluorescent protein, γ-aminobutyrate, lycopene) were improved significantly in the sugar transporter mutants., Conclusions: The elimination of sugar transporter resulted in alteration of global gene expression and redirection of carbon flux distribution, which was purposed to increase energy yield and recycle carbon sources. When the pathways for several valuable compounds were introduced to mutant strains, specific yield of them were highly improved. These results showed that controlling the sugar uptake rate is a good strategy for ameliorating metabolite production.

Depression is the most frequent psychiatric disorder in the world. Recent evidence has shown that stress-induced GABAergic dysfunction in the nucleus accumbens (NAc) contributed to the pathophysiology of depression. However, the molecular mechanisms underlying these pathological changes remain unclear. In this study, mice were constantly treated with the chronic unpredictable mild stress (CUMS) till showing depression-like behaviours expression. GABA synthesis, release and uptake in the NAc tissue were assessed by analysing the expression level of genes and proteins of Gad-1, VGAT and GAT-3 by qRT-PCR and Western blotting. The miRNA/mRNA network regulating GABA was constructed based on the bioinformatics prediction software and further validated by dual-luciferase reporter assay in vitro and qRT-PCR in vivo, respectively. Our results showed that the expression level of GAT-3, Gad-1 and VGAT mRNA and protein significantly decreased in the NAc tissue from CUMS-induced depression-like mice than that of control mice. However, miRNA-144-3p, miRNA-879-5p, miR-15b-5p and miRNA-582-5p that directly down-regulated the expression of Gad-1, VGAT and GAT-3 were increased. In the mRNA/miRNA regulatory GABA network, Gad-1 and VGAT were directly regulated by binding seed sequence of miR-144-3p, and miR-15b-5p, miR-879-5p could be served negative post-regulators by binding to the different sites of VGAT 3'-UTR. Chronic stress causes the impaired GABA synthesis, release and uptake by up-regulating miRNAs and down-regulating mRNAs and proteins, which may reveal the molecular mechanisms for the decreased GABA concentrations in the NAc tissue of CUMS-induced depression., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Polyphenols and γ-aminobutyric acid (GABA) accumulate during seed germination, but the mechanisms involved are poorly understood. The objective of this study was to elucidate the accumulation of these bioactive compounds in Chinese wild rice during germination. The greatest differences in the phenolic content were at 36-h (G36) and 120-h germination (G120) stages. An iTRAQ-based proteomic analysis revealed 7031 proteins, and a comparison of the G120 and G36 stages revealed 956 upregulated and 188 downregulated proteins. The KEGG analysis revealed significant protein enrichment in the "metabolic pathways", "biosynthesis of secondary metabolites" and "phenylpropanoid biosynthesis". Four phenylalanine ammonia-lyases, one 4-coumarate-CoA ligase, one cinnamoyl-CoA reductase, two cinnamyl alcohol dehydrogenases, and four glutamate decarboxylases exhibited higher expression at the G120 than at the G36 stage and promoted phenolics and GABA accumulation. This study revealed bioactive compound accumulation in germinating Chinese wild rice, and the finding may help develop functional foods derived from this cereal., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

γ-Aminobutyrate (GABA) is an important chemical in pharmaceutical field. The use of lactic acid bacteria as biocatalysts for the conversion of l-monosodium glutamate (MSG) into GABA opens interesting perspectives for the production of this functional compound. In this work, an engineered GABA high-producing strain Lactobacillus brevis GadA ΔC14 was constructed by overexpressing a C-terminally truncated GadA mutant, which is active in expanded pH range. After comparison with agar and κ-carrageenan, gellan gum was selected as the optimal immobilization support, and the properties of L. brevis GadA ΔC14  cells encapsulated in this hydrogel were examined. The optimum pH and temperature of immobilized cells were found to be 40°C and pH 4.4, respectively. It was also observed that operational and thermal stabilities of the cells were increased with immobilization. After ten consecutive reaction cycles, the total amounts of GABA produced by the immobilized cells summed up to 87.56 g/L under the optimum experimental conditions. Taken together, the improved stability and good usability make the immobilized L. brevis GadA ΔC14  cells more valuable for industrial applications., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Gamma-aminobutyric acid (GABA)-producing strains were isolated from four edible insects and subjected to 16S rRNA sequence analysis. Among the four GABA-producing bacteria, Enterococcus avium JS-N6B4 exhibited the highest GABA-production, while cultivation temperature, initial pH, aerobic condition, and mono-sodium glutamate (MSG) feeding were found to be the key factors affecting GABA production rate. The culture condition was optimized in terms of glucose, yeast extract, and MSG concentrations using response surface methodology (RSM). GABA production up to 16.64 g/l was obtained under the conditions of 7 g/l glucose, 45 g/l yeast extract, and 62 g/l MSG through the optimization of medium composition by RSM. Experimental GABA production was 13.68 g/l, which was close to the predicted value (16.64 g/l) calculated from the analysis of variance, and 2.79-fold higher than the production achieved with basic medium. Therefore, GABA-producing strains may help improve the GABA production in edible insects, and provide a new approach to the use of edible insects as effective food biomaterials.

Background: In lactic acid bacteria (LAB), acid stress leads to decreases of cell vitality and fermentation yield. Glutamate decarboxylase (GAD) system is regarded as one of the essential acid-resistance mechanisms in LAB. However, the regulation of GAD system is not well identified in the genus Lactobacillus. Although potential transcriptional regulator gene located upstream of GAD system genes was found in several Lactobacillus species, such as Lactobacillus (L.) brevis, the contribution of the regulator to acid resistance of the genus Lactobacillus has not been experimentally determined., Results: The potential transcriptional regulator gene gadR was disrupted by homologous recombination in L. brevis ATCC 367, leading to the decreased expression of gadC and gadB. The inactivation of GadR completely eliminated γ-aminobutyric acid (GABA) production and decreased the glutamate-dependent acid resistance. Moreover, expression of gadC and gadB in the presence of glutamate was increased and glutamate also stimulated the expression of gadR. In addition, L. brevis D17, a strain screened from acidic fermented grains of Chinese liquor production, had much higher expression level of gadR than the typical strain L. brevis ATCC 367. Under the pH-controlled and mixed-feed fermentation, L. brevis D17 achieved a titer of 177.74 g/L and a productivity of 4.94 g/L/h of GABA within 36 h. However, the L. brevis ATCC 367 only achieved a titer of 6.44 g/L and 0.18 g/L/h of GABA although the same fermentation control approach was employed., Conclusions: GadR is a positive transcriptional regulator controlling GABA conversion and acid resistance in L. brevis. L. brevis strains with hyper-expressing of gadR are excellent candidates for GABA production in industrial scale.

Cognitive dysfunction occurs frequently in multiple sclerosis (MS). Research suggests that hippocampal lesions and GABAergic neurotransmitter changes contribute to cognitive dysfunction. In the present study, we aim to determine the cellular changes in GABAergic expression in MS hippocampus related to inflammation and demyelination. To this end, the presence and inflammatory activity of demyelinating lesions was determined by immunohistochemistry in human postmortem hippocampal tissue of 15 MS patients and 9 control subjects. Subsequently, GABAergic cells were visualized using parvalbumin (PV) and glutamate acid decarboxylase 67 (GAD67) markers. Fluorescent colabeling was performed of GAD67 with neuronal nuclei, PV, astrocytic glial fibrillary acidic protein, or vesicular GABA transporter. We observed increased GAD67-positive (GAD67+) neuron and synapse numbers in the CA1 of MS patients with active hippocampal lesions, not due to neurogenesis. The number and size of PV-positive neurons remained unchanged. GAD67+ astrocytes were more numerous in hippocampal white matter than grey matter lesions. Additionally, in MS patients with active hippocampal lesions GAD67+ astrocyte surface area was increased. Disturbed cognition was most prevalent in MS patients with active hippocampal lesions. Summarizing, increased GAD67 immunoreactivity occurs in neurons and astrocytes and relates to hippocampal inflammation and possibly disturbed cognition in MS., (© 2019 American Association of Neuropathologists, Inc.)