Glutamate Decarboxylase Gene Research Articles

Transcriptomics reveal different metabolic strategies for acid resistance and gamma-aminobutyric acid (GABA) production in select Levilactobacillus brevis strains

BackgroundOf the many neurotransmitters in humans, gamma-aminobutyric acid (GABA) shows potential for improving several mental health indications such as stress and anxiety. The microbiota-gut-brain axis is an important pathway for GABAergic effects, as microbially-secreted GABA within the gut can affect host mental health outcomes. Understanding the molecular characteristics of GABA production by microbes within the gut can offer insight to novel therapies for mental health.ResultsThree strains of Levilactobacillus brevis with syntenous glutamate decarboxylase (GAD) operons were evaluated for overall growth, glutamate utilization, and GABA production in typical synthetic growth media supplemented with monosodium glutamate (MSG). Levilactobacillus brevis Lbr-6108™ (Lbr-6108), formerly known as L. brevis DPC 6108, and Levilactobacillus brevis Lbr-35 ™ (Lbr-35) had similar growth profiles but differed significantly in GABA secretion and acid resistance. Lbr-6108 produced GABA early within the growth phase and produced significantly more GABA than Lbr-35 and the type strain Levilactobacillus brevis ATCC 14869 after the stationary phase. The global gene expression during GABA production at several timepoints was determined by RNA sequencing. The GAD operon, responsible for GABA production and secretion, activated in Lbr-6108 after only 6 h of fermentation and continued throughout the stationary phase. Furthermore, Lbr-6108 activated many different acid resistance mechanisms concurrently, which contribute to acid tolerance and energy production. In contrast, Lbr-35, which has a genetically similar GAD operon, including two copies of the GAD gene, showed no upregulation of the GAD operon, even when cultured with MSG.ConclusionsThis study is the first to evaluate whole transcriptome changes in Levilactobacillus brevis during GABA production in different growth phases. The concurrent expression of multiple acid-resistance mechanisms reveals niche-specific metabolic functionality between common human commensals and highlights the complex regulation of GABA metabolism in this important microbial species. Furthermore, the increased and rapid GABA production of Lbr-6108 highlights the strain’s potential as a therapeutic and the overall value of screening microbes for effector molecule output.

Characterization of three glutamate decarboxylases from Bacillus spp. for efficient \u03b3-aminobutyric acid production

BackgroundGamma-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.ResultsThree 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.ConclusionsThree 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.

Characterization and comparative genomic analysis of gamma-aminobutyric acid (GABA)-producing lactic acid bacteria from Thai fermented foods.

This study aimed to screen, characterize, and annotate the genome along with the comparison of GABA synthesis genes presented in lactic acid bacteria (LAB). Thirty-five LAB isolates from fermented foods were screened for GABA production using thin-layer chromatography (TLC). Fifteen isolates produced GABA ranging from 0.07 to 22.94 g/L. Based on their GTG5 profiles, phenotypic, and genotypic characteristics, isolates LSI1-1, LSI1-5, LSI2-1, LSI2-2, LSI2-3, LSI2-5, and LSM3-1-4 were identified as Lactobacillus plantarum subsp. plantarum; isolate LSM1-4 was Lactobacillus argentoratensis; isolates CAB1-2, CAB1-5, CAB1-7, and LSI1-4 were Lactobacillus pentosus; and CAB1-1, LSM3-1-1 and LSM3-2-3 were Lactobacillus fermentum. Strains LSI2-1 and CAB1-7 from pickled vegetables were selected for genome analysis. The gadA gene (1410 bp, 470aa) was encountered in GABA production of both strains and no other glutamate decarboxylase (GAD) genes were found in the genomes when compared with other LAB strains. The presence of gadA is evidence for GABA production. Strains LSI2-1 and CAB1-7 produced 22.94 g/L and 11.59 g/L of GABA in GYP broth supplemented with 3% (w/v) MSG at 30 °C for 72 h, respectively. Our report highlights the characterization of LAB and GABA production of L. plantarum LSI2-1 strain with its GABA synthesis gene. GABA production of strains LSI2-1 and CAB1-7 in GYP broth with 3% (w/v) MSG and comparative GAD genes.

Exogenous application of GABA retards cap browning in Agaricus bisporus and its possible mechanism

In this study, the effect of exogenous γ-aminobutyric acid (GABA) at 0 (control), 0.01, 0.1, 1, and 10 mM on cap browning and quality of button mushrooms during 15 d of storage at 4 °C was investigated. GABA at 0.1 mM yielded the lowest cap browning. Weight loss, electrolyte leakage, and malondialdehyde (MDA) content were significantly lower in mushrooms treated with 0.1 mM GABA, while their firmness was higher. The increased accumulation of total phenolic compounds in the 0.1 mM GABA treated mushrooms was associated with a higher expression and activity of phenylalanine ammonia-lyase (PAL) accompanied by a lower expression and activity of polyphenol oxidase (PPO). GABA treatment was capable of reducing H2O2 content and increasing ascorbic acid (AsA) content. Exogenous GABA increased the endogenous GABA content by increasing the expression of the glutamate decarboxylase (GAD) gene and decreasing the expression of the GABA transaminase (GABA-T) gene. Overall, GABA treatment could be employed for retarding cap browning and maintaining the sensory and nutritional quality of button mushrooms during cold storage.

Genome analysis and optimization of γ-aminobutyric acid (GABA) production by lactic acid bacteria from plant materials.

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 13768T. 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.

Subchronic neurotoxicity of diazinon in albino mice: Impact of oxidative stress, AChE activity, and gene expression disturbances in the cerebral cortex and hippocampus on mood, spatial learning, and memory function.

Diazinon (DZN) with prominent neurotoxic effects perturbs CNS function via multiple mechanisms. This investigation intends to explore mood, spatial learning, and memory dysfunction, acetylcholine esterase (AChE) activity, and neurodegeneration-related gene expression in the cortex and hippocampus regions of mice exposed to DZN for 63 consecutive days (subchronic exposure). Adult male albino mice were orally given sublethal DZN (DZNL = 0.1 mg/kg, DZNM = 1 mg/kg and DZNH = 10 mg/kg). All mice in the DZNH group died within 3 weeks postexposure. DZNL and DZNM caused body and brain weight loss (p < 0.05). Completing 9 weeks of DZN exposure, a marked decline in AChE activity and oxidative stress level was indicated in both brain regions (p < 0.05). Also, synaptophysin, vesicular acetylcholine transferase, and glutamate decarboxylase gene expressions were affected in both brain regions (p < 0.05). Furthermore, the present study revealed that DZN administration increased anxiety and depressive-like behaviors (p < 0.0001). Spatial learning and short- and long-memory were severely affected by DZNL and DZNM treatments (p < 0.0001). Taken together, subchronic exposure to low and medium doses of DZN can cause AChE inhibition, oxidative damage, and neurotransmitter disturbances in brain cells and induce neurodegeneration. These changes would impair mood, spatial learning, and memory function.

γ-Aminobutyric Acid Suppresses Iron Transportation from Roots to Shoots in Rice Seedlings by Inducing Aerenchyma Formation.

γ-Aminobutyric acid (GABA) is a widely distributed non-protein amino acid mediated the regulation of nitrate uptake and Al3+ tolerance in plants. However, there are few reports about the involvement of GABA in the regulation of iron (Fe) acquisition and translocation. Here, we show that GABA regulates Fe homeostasis in rice seedlings. Exogenous GABA decreased the chlorophyll concentration in leaves, with or without Fe supply. Over-expression of glutamate decarboxylase (GAD) gene, coding a crucial enzyme of GABA production, elevated endogenous GABA content and caused more leaf chlorosis than wild type (Nipponbare). GABA inhibited Fe transportation from roots to shoots and GABA application elevated the expression levels of Fe deficiency (FD)-related genes under conditions of Fe-sufficiency (FS), suggesting that GABA is a regulator of Fe translocation. Using Perls’ blue staining, we found that more ferric iron (Fe3+) was deposited in the epidermal cells of roots treated with GABA compared with control roots. Anatomic section analysis showed that GABA treatment induced more aerenchyma formation compared with the control. Aerenchyma facilitated the oxidization of soluble ferrous iron (Fe2+) into insoluble Fe3+, resulted in Fe precipitation in the epidermis, and inhibited the transportation of Fe from roots to shoots.

The influence of laser beam and high light intensity on lentil (Lens culinaris) and wheat (Triticum aestivum) seedlings growth and metabolism

Light is considered as an environmental signal that controls plants growth and development. Sufficient duration, quality and wavelength are mainly responsible for switching on/off many physiological processes. The aim of this study was to examine the role of gamma aminobutyric acid (GABA) shunt pathway specific response in wheat (Triticum aestivum) and lentil (Lens culinaris) seedlings to blue LED laser (intensity= 1.48 W/cm2) and high intensity invisible ultraviolet light (intensity= 3.99 W/cm2) treatments separately, with respect to: seed germination, seedling growth, oxidative damage in term of reactive oxygen substances accumulation, GABA accumulation level, total proteins and total carbohydrates level and glutamate decarboxylase gene (GAD) expression. Data showed a remarkable changes in proteins content, carbohydrates content, chlorophyll level, dry and fresh weight, GABA accumulation, MDA level, and GAD m-RNA level under blue LED laser and high intensity invisible ultraviolet light treatments; separately in the both wheat and lentil cultivars. There were no significant differences of root emergence in all cultivars under all treatments. The concentrations of MDA increased in all cultivars under blue LED laser and high intensity invisible ultraviolet light treatments with increasing exposure durations. GABA levels increased and correlated significantly in all cultivars as the exposure duration of blue LED laser and high light intensity increased. Significant decrease in chlorophyll a and b contents in Um Qayes, Jordan1 and Jordan2 cultivars under all laser and high intensity invisible ultraviolet light treatments. A significant difference was found in proteins content in all cultivars in response to laser and high intensity invisible ultraviolet light treatments. Carbohydrates content in all cultivars used in this study were significantly decreased under laser treatments. The transcription level of GAD increased significantly under blue LED laser and high intensity invisible ultraviolet light in all cultivars used in this study. GAD expression was high in Hurani75 cultivar in agreement with the low level of GABA accumulation under high intensity invisible ultraviolet light at 60 min which strongly suggested that GABA was catabolized to enter another pathways (C:N balance) that was confirmed by increase in higher level of carbohydrates and proteins beside reduction in MDA content at the same exposure duration. On other hand, GAD expression in Jordan 2 was lower in response to laser treatment under the same exposure duration. The high increase of GAD gene expression explains the high accumulation of GABA level under both treatments. Results indicated that GABA shunt is a key metabolic pathway that allows wheat and lentil to adapt blue LED laser and high intensity invisible ultraviolet light stressful treatments.

Gamma-aminobutyric acid (GABA) alleviates salt damage in tomato by modulating Na+ uptake, the GAD gene, amino acid synthesis and reactive oxygen species metabolism

BackgroundSalt stress is a serious abiotic stress that caused crop growth inhibition and yield decline. Previous studies have reported on the the synthesis of gamma-aminobutyric acid (GABA) and its relationship with plant resistance under various abiotic stress. However, the relationship between exogenous GABA alleviating plant salt stress damage and ion flux, amino acid synthesis, and key enzyme expression remains largely unclear. We investigated plant growth, Na+ transportation and accumulation, reactive oxygen species (ROS) metabolism and evaluated the effect of GABA on amino acids, especially SlGADs gene expression and the endogenous GABA content of tomato (Solanum lycopersicum L.) seedlings treated with or without 5 mmol·L− 1 GABA under 175 mmol·L− 1 NaCl stress.ResultsExogenous application of GABA significantly reduced the salt damage index and increased plant height, chlorophyll content and the dry and fresh weights of tomato plants exposed to NaCl stress. GABA significantly reduced Na+ accumulation in leaves and roots by preventing Na+ influx in roots and transportation to leaves. The transcriptional expression of SlGAD1–3 genes were induced by NaCl stress especially with GABA application. Among them, SlGAD1 expression was the most sensitive and contributed the most to the increase in glutamate decarboxylase (GAD) activity induced by NaCl and GABA application; Exogenous GABA increased GAD activity and amino acid contents in tomato leaves compared with the levels under NaCl stress alone, especially the levels of endogenous GABA, proline, glutamate and eight other amino acids. These results indicated that SlGADs transcriptional expression played an important role in tomato plant resistance to NaCl stress with GABA application by enhancing GAD activity and amino acid contents. GABA significantly alleviated the active oxygen-related injury of leaves under NaCl stress by increasing the activities of antioxidant enzymes and decreasing the contents of active oxygen species and malondialdehyde.ConclusionExogenous GABA had a positive effect on the resistance of tomato seedlings to salt stress, which was closely associated with reducing Na+ flux from root to leaves, increasing amino acid content and strengthening antioxidant metabolism. Endogenous GABA content was induced by salt and exogenous GABA at both the transcriptional and metabolic levels.

Crosstalk between GABA and ALA to improve antioxidation and cell expansion of tomato seedling under cold stress

Cold stress inhibits plant growth and ultimately affects yield formation. Exogenous 5- aminolevulinic acid (ALA) can improve tomato cold tolerance and promote plant growth. It significantly up-regulated glutamate decarboxylase gene (SlGAD4) expression, which was involved in gamma-aminobutyric acid (GABA) synthesis. Whether the GABA was involved in ALA-regulated tomato cold tolerance and plant growth was unclear. Thus, this study aimed to explore the effects of exogenous ALA and GABA on endogenous ALA and GABA synthesis. The roles of ALA and GABA on regulating the antioxidation and cell morphological changes related to plant growth in tomato leaves were further explored. And the internal relationship between ALA and GABA in increasing tomato cold tolerance was also determined. Results showed that cold stress increased the glutamate (Glu) and GABA contents and reduced the ALA content. The exogenous ALA- or GABA-treated plants demonstrated decreased Glu content and increased GABA or ALA contents. Exogenous ALA or GABA treatment promoted the expansion of upper epidermal (UEP) and palisade parenchymal (PA) cells and up-regulated the xyloglucan endotransglucosylase/hydrolases gene (SlXTH23) expression in tomato leaves. Exogenous ALA or GABA significantly alleviated cold-induced tomato membrane lipid peroxidation and enhanced C-repeat binding factors gene (SlCBF2) expression and superoxide dismutase (SOD), catalase (CAT), APX and GR activities. Inhibiting endogenous GABA with 3-mercaptopropionic (3-mp) dramatically decreased the size of UEP and PA cells and reduced SOD and CAT activities. It also aggravated the membrane peroxidation damage, which notably weakened the alleviated effects of exogenous ALA. Inhibiting endogenous ALA with gabaculine (gaba) partly inhibited the alleviated effects of exogenous GABA. These findings indicated that spray with exogenous ALA or GABA could increase endogenous ALA and GABA levels in tomato leaves. In addition, endogenous GABA and ALA could be mutually converted through Glu. Exogenous ALA and GABA promoted cell expansion, stimulated the antioxidant system and might enhance tomato cold tolerance via endogenous GABA signal and the C-repeat binding factor regulation pathway, thereby alleviating cold inhibited plant growth. In conclusion, GABA crosstalk with ALA improved tomato cold stress tolerance and promoted plant growth by regulating antioxidants and cell expansion.

The involvement of gamma-aminobutyric acid shunt in the endoplasmic reticulum stress response of Arabidopsis thaliana

The endoplasmic reticulum (ER) is the main site of secretory protein production and folding and its homeostasis under environmental stress is vital for the maintenance of the protein secretory pathway. The loss of homeostasis and accumulation of unfolded proteins in the ER is referred to as ER stress. Although, γ-aminobutyric acid (GABA) is an important regulator of stress response in plants, its roles during ER stress remains unclear. This study investigated the involvement of GABA in the ER stress response of plants. For this, changes in GABA metabolism under ER stress was analysed in Arabidopsis thaliana, then to study the response of the ER-folding machinery, plants were treated with exogenous GABA under ER stress. The antibiotic tunicamycin, which inhibits N-glycosylation was used to specifically induce ER stress. This stress up-regulated the expression of five glutamate decarboxylase (GAD) genes except GAD2 and GABA content of A. thaliana plants increased with an increasing concentration of tunicamycin (0.1 μg ml−1 and 0.25 μg ml−1). Moreover, expressions of genes involved in the conversion of GABA to succinate was also induced, while genes involved in transport across plasma and mitochondrial membrane showed no response to ER stress. The exogenous treatment of plants with 1-and 5-mM GABA increased plant performance under ER stress but 0.1 mM proved ineffective. Plants treated with GABA under ER stress had decreased expression of ER stress marker genes such as BIP1, BIP3 or CNX, but the expression of genes related to ER stress perception or ER-associated protein degradation showed no changes with respect to GABA treatments.

Induction of γ-aminobutyric acid plays a positive role to Arabidopsis resistance against Pseudomonas syringae.

Gamma‐aminobutyric acid (GABA) is an important metabolite which functions in plant growth, development, and stress responses. However, its role in plant defense and how it is regulated are largely unknown. Here, we report a detailed analysis of GABA induction during the resistance response to Pseudomonas syringae in Arabidopsis thaliana. While searching for the mechanism underlying the pathogen‐responsive mitogen‐activated protein kinase (MPK)3/MPK6 signaling cascade in plant immunity, we found that activation of MPK3/MPK6 greatly induced GABA biosynthesis, which is dependent on the glutamate decarboxylase genes GAD1 and GAD4. Inoculation with Pseudomonas syringae pv tomato DC3000 (Pst) and Pst‐avrRpt2 expressing the avrRpt2 effector gene induced GAD1 and GAD4 gene expression and increased the levels of GABA. Genetic evidence revealed that GAD1, GAD2, and GAD4 play important roles in both GABA biosynthesis and plant resistance in response to Pst‐avrRpt2 infection. The gad1/2/4 triple and gad1/2/4/5 quadruple mutants, in which the GABA levels were extremely low, were more susceptible to both Pst and Pst‐avrRpt2. Functional loss of MPK3/MPK6, or their upstream MKK4/MKK5, or their downstream substrate WRKY33 suppressed the induction of GAD1 and GAD4 expression after Pst‐avrRpt2 treatment. Our findings shed light on both the regulation and role of GABA in the plant immunity to a bacterial pathogen.

Expression of two glutamate decarboxylase genes in Lactobacillus brevis during gamma-aminobutyric acid production with date residue extract.

Gamma-aminobutyric acid (GABA) is produced by Lactobacillus brevis using date residue fermentation. In this study, the GABA production method was improved, for which L. brevis strain JCM 1059T was the most efficient among the four L. brevis strains examined. This was presumably due to a difference in the expression level of the gene encoding glutamate decarboxylase that catalyzes GABA synthesis.Abbreviation: GABA: gamma-aminobutyric acid.

An In Vivo Targeted Deletion of the Calmodulin-Binding Domain from Rice Glutamate Decarboxylase 3 (OsGAD3) Increases \u03b3-Aminobutyric Acid Content in Grains

BackgroundGamma-aminobutyric acid (GABA) is a non-protein amino acid present in all living things. GABA is mainly synthesized from glutamate by glutamate decarboxylase (GAD). In plants the enzymatic activity of GAD is activated by Ca2+/calmodulin binding (CaMBD) at the C-terminus in response to various stresses, allowing rapid GABA accumulation in cells. GABA plays a central role in not only stress responses but also many aspects of plant growth and development as a signaling molecules. Furthermore, it is known to be a health-promoting functional substance that exerts improvements in life-style related diseases such as hypertension, diabetes, hyperlipidemia, and so on. Previous reports indicated that CaMBD found plant GADs possess an autoinhibitory function because truncation of GAD resulted in extreme GABA accumulation in plant cells. Therefore, we attempted a genetic modification of rice GAD via genome editing technology to increase GABA levels in the edible part of rice.ResultsIn this study, we focused on GAD3, one of five GAD genes present in the rice genome, because GAD3 is the predominantly expressed in seeds, as reported previously. We confirmed that GAD3 has an authentic Ca2+/CaMBD that functions as an autoinhibitory domain. CRISPR/Cas9-mediated genome editing was performed to trim the coding region of CaMBD off from the OsGAD3 gene, then introducing this transgene into rice scutellum-derived calli using an all-in-one vector harboring guide RNAs and CRISPR/Cas9 via Agrobacterium to regenerate rice plants. Out of 24 transformed rice (T1), a genome-edited rice line (#8_8) derived from two independent cleavages and ligations in the N-terminal position encoding OsGAD3-CaMBD and 40 bp downstream of the termination codon, respectively, displayed a AKNQDAAD peptide in the C-terminal region of the putative OsGAD3 in place of its intact CaMBD (bold indicates the trace of the N-terminal dipeptides of the authentic CaMBD). A very similar rice line (#8_1) carrying AKNRSSRRSGR in OsGAD3 was obtained from one base pair deletion in the N-terminal coding region of the CaMBD. Free amino acid analysis of the seeds (T2) indicated that the former line contained seven-fold higher levels of GABA than wild-type, whereas the latter line had similar levels to the wild-type, although in vitro enzyme activities of recombinant GAD proteins based on the GAD3 amino acid sequence elucidated from these two lines in the absence of Ca2+/bovine CaM were both higher than wild-type counterpart. In addition to high level of GABA in #8_8, the average seed weight per grain and protein content were superior to wild-type and #8_1.ConclusionsWe have successfully established GABA-fortified rice by using CRISPR/Cas9 genome editing technology. Modified rice contained seven-fold higher GABA content and furthermore displayed significantly higher grain weight and protein content than wild-type brown rice. This is the first report of the production of GABA-enriched rice via a genome editing.

Cytotoxic Evaluation of Antioxidant and Gamma-Amino Butyric Acid (GABA) Enriched Mango (Mangifera indica cv. Chaunsa) Mesocarp Extract on HER2-Positive Breast Cancer Cells

Mango (Mangifera indica L. cv. Chaunsa Sumer bahist) fruit is renowned for its flavor and aroma. Besides, it had been found to have large antioxidant content as well as anti-cancer and anti-diabetic properties. However, experiments to test both the antioxidant content and health benefits are still needed. Here, in the ripe mango mesocarp, we have measured the expression of 11 genes playing a role in flavor and aroma development. An up-regulation in expression of fructose-bisphosphate_aldolase (MiFBA6), glutamate decarboxylase (MiGAD), catalase (MiCAT1), glutathione-S-transferase (MiGSTF6) and nucleoredoxin (MiNRX1) genes was recorded. MiGAD gene expression indicated the presence of γ-aminobutyric acid (GABA) metabolite in mesocarp tissue of Chaunsa mango. Metabolite profiling was carried out by gas chromatography-mass spectrometry. A total of five antioxidant compounds with known anticancer activities were characterized in mango mesocarp. It was found metabolites belong to various chemical groups such as: monoterpene alcohols, aroma volatiles, glycoside, monosaccharides, disaccharides, trisaccharides, oligosaccharides, sugar alcohols, sugar acids, essential oils, amino acids, ketone bodies, phytohormones and fatty acids. Seven antioxidant metabolites with known anticancer activities were characterized in mango mesocarp including: 3-hydroxybutyric acid, linalool, geraniol, erythritol, L-(+)-tartaric acid, myo-inositol and lactitol. A total of eight distinct aroma generating siloxanes were characterized in this cultivar. Positive antioxidant activity was also observed in the ethanolic extract of Chaunsa fruit pulp. Chemotherapeutic potential of phenolic compounds found in the ethyl acetate (EtOAc) extract on human breast cancer cell line AU565 was also investigated. The EtOAc extracts effectively suppressed adenocarcinoma growth indicating the anticancer potential of Chaunsa mango fruit.

Cytotoxic Evaluation of Antioxidant and Gamma-Amino Butyric Acid (GABA) Enriched Mango (Mangifera indica cv. Chaunsa) Mesocarp Extract on HER2-Positive Breast Cancer Cells

Mango (Mangifera indica L. cv. Chaunsa Sumer bahist) fruit is renowned for its flavor and aroma. Besides, it had been found to have large antioxidant content as well as anti-cancer and anti-diabetic properties. However, experiments to test both the antioxidant content and health benefits are still needed. Here, in the ripe mango mesocarp, we have measured the expression of 11 genes playing a role in flavor and aroma development. An up-regulation in expression of fructose-bisphosphate_aldolase (MiFBA6), glutamate decarboxylase (MiGAD), catalase (MiCAT1), glutathione-S-transferase (MiGSTF6) and nucleoredoxin (MiNRX1) genes was recorded. MiGAD gene expression indicated the presence of γ-aminobutyric acid (GABA) metabolite in mesocarp tissue of Chaunsa mango. Metabolite profiling was carried out by gas chromatography-mass spectrometry. A total of five antioxidant compounds with known anticancer activities were characterized in mango mesocarp. It was found metabolites belong to various chemical groups such as: monoterpene alcohols, aroma volatiles, glycoside, monosaccharides, disaccharides, trisaccharides, oligosaccharides, sugar alcohols, sugar acids, essential oils, amino acids, ketone bodies, phytohormones and fatty acids. Seven antioxidant metabolites with known anticancer activities were characterized in mango mesocarp including: 3-hydroxybutyric acid, linalool, geraniol, erythritol, L-(+)-tartaric acid, myo-inositol and lactitol. A total of eight distinct aroma generating siloxanes were characterized in this cultivar. Positive antioxidant activity was also observed in the ethanolic extract of Chaunsa fruit pulp. Chemotherapeutic potential of phenolic compounds found in the ethyl acetate (EtOAc) extract on human breast cancer cell line AU565 was also investigated. The EtOAc extracts effectively suppressed adenocarcinoma growth indicating the anticancer potential of Chaunsa mango fruit.

GABA synthesis mediated by γ-aminobutanal dehydrogenase in Synechocystis sp. PCC6803 with disrupted glutamate and α-ketoglutarate decarboxylase genes

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.

Metagenomic Insights Into the Taxonomic and Functional Features of Kinema, a Traditional Fermented Soybean Product of Sikkim Himalaya.

Kinema is an ethnic, naturally fermented soybean product consumed in the Sikkim Himalayan region of India. In the present study, the whole metagenome sequencing approach was adopted to examine the microbial diversity and related functional potential of Kinema, consumed in different seasons. Firmicutes was the abundant phylum in Kinema, ranging from 82.31 to 93.99% in different seasons, followed by Actinobacteria and Proteobacteria. At the species level, the prevalent microorganisms were Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Corynebacterium glutamicum, Bacillus pumilus, and Lactococcus lactis. The abundance of microbial species varied significantly in different seasons. Further, the genomic presence of some undesirable microbes like Bacillus cereus, Proteus mirabilis, Staphylococcus aureus, Proteus penneri, Enterococcus faecalis, and Staphylococcus saprophyticus, were also detected in the specific season. The metagenomic analysis also revealed the existence of bacteriophages belonging to the family Siphoviridae, Myoviridae, and Podoviridae. Examination of the metabolic potential of the Kinema metagenome depicted information about the biocatalysts, presumably involved in the transformation of protein and carbohydrate polymers into bioactive molecules of health-beneficial effects. The genomic resource of several desirable enzymes was identified, such as β-galactosidase, β-glucosidase, β-xylosidase, and glutamate decarboxylase, etc. The catalytic function of a novel glutamate decarboxylase gene was validated for the biosynthesis of γ-aminobutyric acid (GABA). The results of the present study highlight the microbial and genomic resources associated with Kinema, and its importance in functional food industry.

Disruption of cyanobacterial \u03b3-aminobutyric acid shunt pathway reduces metabolites levels in tricarboxylic acid cycle, but enhances pyruvate and poly(3-hydroxybutyrate) accumulation

The photoautotrophic cyanobacterium Synechocystis sp. PCC 6803 assimilates carbon dioxide as the sole carbon source, and a major portion of the assimilated carbon is metabolically consumed by the tricarboxylic acid (TCA) cycle. Effects of partial interference of TCA cycle metabolic activity on other carbon metabolism have yet to be examined. Here, the γ-aminobutyric acid (GABA) shunt, one of the metabolic pathways for completing TCA cycle in Synechocystis, was disrupted via inactivating the glutamate decarboxylase gene (gdc). Under normal photoautotrophic condition, cell growth and the level of the TCA cycle metabolites succinate, malate and citrate were decreased by 25%, 35%, 19% and 28%, respectively, in Δgdc mutant relative to those in the wild type (WT). The cellular levels of glycogen and total lipids of the Δgdc mutant were comparable to those of the WT, but the intracellular levels of pyruvate and bioplastic poly(3-hydroxybutyrate) (PHB) were 1.23- and 2.50-fold higher, respectively, in Δgdc mutant. Thus, disruption of the GABA shunt pathway reduced the TCA cycle metabolites levels, but positively enhanced the bioaccumulation of pyruvate and PHB. The PHB production rate in Δgdc mutant was 2.0-fold higher than in the WT under normal photoautotrophy.

Identification, Expression and Preliminary Characterization of a Recombinant Bifunctional Enzyme of Photobacterium damselae subsp. piscicida with Glutamate Decarboxylase/Transaminase Activity

Glutamate decarboxylase catalyzes the conversion of glutamate to gamma-aminobutyric acid (GABA), contributing to pH homeostasis through proton consumption. The reaction is the first step toward the GABA shunt. To date, the enzymes involved in the glutamate metabolism of Photobacterium damselae subsp. piscicida have not been elucidated. In this study, an open reading frame of P. damselae subsp. piscicida, showing homology to the glutamate decarboxylase or putative pyridoxal-dependent aspartate 1-decarboxylase genes, was isolated and cloned into an expression vector to produce the recombinant enzyme. Preliminary gas chromatography-mass spectrometry characterization of the purified recombinant enzyme revealed that it catalyzed not only the decarboxylation of glutamate but also the transamination of GABA. This enzyme of P. damselae subsp. piscicida could be bifunctional, combining decarboxylase and transaminase activities in a single polypeptide chain.