B cell-derived GABA elicits IL-10+ macrophages to limit anti-tumour immunity.

Small, soluble metabolites not only are essential intermediates in intracellular biochemical processes, but can also influence neighbouring cells when released into the extracellular milieu1-3. 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.

Genetically engineered Lactococcus lactis strain constitutively expresses GABA-producing genes and produces high levels of GABA.

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.

Optimization of fermentation conditions for the production of γ-aminobutyric acid by Lactobacillus hilgardii GZ2 from traditional Chinese fermented beverage system.

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.

Pyridoxal kinase inhibition by artemisinins down-regulates inhibitory neurotransmission.

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-Šresolution. 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.

Postnatal Development of Glutamate and GABA Transcript Expression in Monkey Visual, Parietal, and Prefrontal Cortices.

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.

Characterization of three glutamate decarboxylases from Bacillus spp. for efficient γ-aminobutyric acid production.

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.

Gut microbiota alterations reveal potential gut-brain axis changes in polycystic ovary syndrome.

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.

Evaluation of gamma-aminobutyric acid (GABA) production by Lactobacillus plantarum using two-step fermentation.

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.

Effects on Metabolism in Astrocytes Caused by cGAMP, Which Imitates the Initial Stage of Brain Metastasis.

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.

Enhancing the production of γ-aminobutyric acid in Escherichia coli BL21 by engineering the enzymes of the regeneration pathway of the coenzyme factor pyridoxal 5'-phosphate.

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).

Immobilization and enzymatic properties of glutamate decarboxylase from Enterococcus faecium by affinity adsorption on regenerated chitin.

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 mgcbd-gad/grc 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 AgNO3, MnSO4, MgSO4, CuSO4, ZnSO4, FeCl2, FeCl3, AlCl3, CaCl2, and Pb(CH3COO)2. The apparent Km and Vmax were 28.35 mmol/L and 147.06 µmol/(gRC-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.

Effect of DR1558, a Deinococcus radiodurans response regulator, on the production of GABA in the recombinant Escherichia coli under low pH conditions.

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·H2O) 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·H2O). 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.

Efficient production of gamma-aminobutyric acid by engineered Saccharomyces cerevisiae with glutamate decarboxylases from Streptomyces.

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 OD600 30, 0.1 M l-Glu, and 0.28 mM pyridoxal phosphate in 0.2 M Na2 HPO4 -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.

A Brief Review on the Non-protein Amino Acid, Gamma-amino Butyric Acid (GABA): Its Production and Role in Microbes.

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.

Dopaminergic modulation of basal ganglia output through coupled excitation-inhibition.

Learning and maintenance of skilled movements require exploration of motor space and selection of appropriate actions. Vocal learning and social context-dependent plasticity in songbirds depend on a basal ganglia circuit, which actively generates vocal variability. Dopamine in the basal ganglia reduces trial-to-trial neural variability when the bird engages in courtship song. Here, we present evidence for a unique, tonically active, excitatory interneuron in the songbird basal ganglia that makes strong synaptic connections onto output pallidal neurons, often linked in time with inhibitory events. Dopamine receptor activity modulates the coupling of these excitatory and inhibitory events in vitro, which results in a dynamic change in the synchrony of a modeled population of basal ganglia output neurons receiving excitatory and inhibitory inputs. The excitatory interneuron thus serves as one biophysical mechanism for the introduction or modulation of neural variability in this circuit.

The molecular mechanism underlying GABAergic dysfunction in nucleus accumbens of depression-like behaviours in mice.

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.

Neural distinctiveness declines with age in auditory cortex and is associated with auditory GABA levels.

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.

Transcriptional profiling aligned with in situ expression image analysis reveals mosaically expressed molecular markers for GABA neuron sub-groups in the ventral tegmental area.

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.

Deciphering the crucial roles of transcriptional regulator GadR on gamma-aminobutyric acid production and acid resistance in Lactobacillus brevis.

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.

Metabolic perturbations in mutants of glucose transporters and their applications in metabolite production in Escherichia coli.

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 13C metabolic flux analysis (13C 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.