Gamma-aminobutyric Acid Accumulation Research Articles

A multi-omics study revealed the effect of pulsed light treatment on germinated brown rice: Promotion of sprouting efficiency and gamma-aminobutyric acid enrichment

Physical fields are increasingly proposed in the production of germinated grain foods, thus shorting the preparation cycle of high value-added sprouted grain foods. Here, pulsed light treatment (PLT) with 300 times at a single energy of 400 J was found to significantly promote the shoot length and gamma-aminobutyric acid (GABA) content in 8 varieties of germinated brown rice (GBR). Functional annotation analysis showed that the brown rice germination was promoted by the biosynthesis of phenylalanine, carbohydrate metabolism, and energy metabolism pathway. Moreover, PLT influenced the genes and metabolites involved in the GABA shunt and the polyamine degradation pathway, promoting the accumulation of GABA. Transcription factor OsbZIP56 was closely related to the transcript level of regulatory gene for germination and GABA enrichment. Furthermore, overexpression of OsbZIP56 in rice significantly promoted germination and GABA accumulation. These findings would be significant for improving the germination efficiency of edible seeds, promoting accumulation of active ingredients, and exploring gene functions in food materials.

The reprogramming of mitochondrial energy metabolism in Rice: The role of the TCA cycle and GABA shunt under Arsenic exposure

AbstractArsenic (As) pollution is hazardous for all living organisms. Plants growing in As‐contaminated soil are less performant. Recently, seed priming has attracted significant attention for its beneficial effects on plants. Numerous studies have associated priming technique with the induction of stress memory against traumatic conditions, but its relation to crop's energy balance is under‐researched. The present study highlights variations in energy metabolic pathways, particularly TCA (tricarboxylic acid) cycle and GABA (gamma‐aminobutyric acid)‐shunt in multi‐nutrient [silicon (Si), iron (Fe), zinc (Zn), and ascorbic acid (AA)] primed rice seedlings under As stress. Arsenic exposure decreased the TCA cycle enzymes' activity while intensifying GABA accumulation and metabolism. This enhancement in GABA shunt provides an alternative energy source for As‐stressed seedlings. However, priming and further Fe‐supplementation reduced As‐toxicity by accelerating the activity of both energy pathways, namely, TCA and GABA cycle. Improved energy regulation by priming and Fe‐augmentation also enhanced the functionality of anabolic respiratory‐interactive‐pathway, i.e., carbon‐nitrogen metabolism. The As‐tolerance response of priming and Fe‐augmentation was further analyzed from the expression profiling of nitrogen and phosphorous utilization genes. Out of 10 genes, particular enhancement was observed in OsNiR, OsGS, and OsGOGAT expression with improved macro‐micronutrient content of the seedlings. Thus, irrespective of As infestations, multi‐nutrient (Si, Fe, Zn, and AA) seed priming approach along with Fe‐supplementation may emerge as a beneficial strategy to improve plant metabolic capacity in terms of nitrogen, phosphorous, and carbon assimilation to support normal vigor because they balance cellular energy levels.

Succinic semialdehyde dehydrogenase deficiency in mice and in humans: An untargeted metabolomics perspective.

Succinic semialdehyde dehydrogenase deficiency (SSADHD) is a rare neurometabolic disorder caused by disruption of the gamma-aminobutyric acid (GABA) pathway. A more detailed understanding of its pathophysiology, beyond the accumulation of GABA and gamma-hydroxybutyric acid (GHB), will increase our understanding of the disease and may support novel therapy development. To this end, we compared biochemical body fluid profiles from SSADHD patients with controls using next-generation metabolic screening (NGMS). Targeted analysis of NGMS data from cerebrospinal fluid (CSF) showed a moderate increase of aspartic acid, glutaric acid, glycolic acid, 4-guanidinobutanoic acid, and 2-hydroxyglutaric acid, and prominent elevations of GHB and 4,5-dihydroxyhexanoic acid (4,5-DHHA) in SSADHD samples. Remarkably, the intensities of 4,5-DHHA and GHB showed a significant positive correlation in control CSF, but not in patient CSF. In an established zebrafish epilepsy model, 4,5-DHHA showed increased mobility that may reflect limited epileptogenesis. Using untargeted metabolomics, we identified 12 features in CSF with high biomarker potential. These had comparable increased fold changes as GHB and 4,5-DHHA. For 10 of these features, a similar increase was found in plasma, urine and/or mouse brain tissue for SSADHD compared to controls. One of these was identified as the novel biomarker 4,5-dihydroxyheptanoic acid. The intensities of selected features in plasma and urine of SSADHD patients positively correlated with the clinical severity score of epilepsy and psychiatric symptoms of those patients, and also showed a high mutual correlation. Our findings provide new insights into the (neuro)metabolic disturbances in SSADHD and give leads for further research concerning SSADHD pathophysiology.

Bacillus amyloliquefaciens Increases the GABA in Rice Seed for Upregulation of Type I Collagen in the Skin

Biosynthesis of gamma-aminobutyric acid (GABA) can be achieved by naturally occurring microorganisms with the advantages of cost-effectiveness and safety. In this study, Bacillus amyloliquefaciens EH-9 strain (B. amyloliquefaciens EH-9), a soil bacterium, was used to promote the accumulation of GABA in germinated rice seed. Further, the topical application of supernatant from rice seed co-cultivated with soil B. amyloliquefaciens EH-9 can significantly increase the production of type I collagen (COL1) in the dorsal skin of mice. The knocking down of the GABA-A receptor (GABAA) significantly reduced the production of COL1 in the NIH/3T3 cells and in the dorsal skin of mice. This result suggests that topical application of GABA can promote the biosynthesis of COL1 via its interaction with the GABAA receptor in the dorsal skin of mice. In summary, our findings illustrate for the first time that soil B. amyloliquefaciens EH-9 elicits GABA production in germinated rice seed to upregulate the formation of COL1 in the dorsal skin of mice. This study is translational because the result shows a potential remedy for skin aging by stimulating COL1 synthesis using biosynthetic GABA associated with B. amyloliquefaciens EH-9.

PS-C23-11: CREATION OF TOMATOES WITH HIGH GABA ACCUMULATION BY GENE EDITING AND THEIR HYPOTENSIVE EFFECTS

Background and Purpose: The number of functional food products containing gamma-aminobutyric acid (GABA), which is claimed to have a hypotensive effect, will exceed 300 in Japan by 2020. GABA containing foods has surpassed the number of applications for non-digestible dextrin, which is claimed to prevent high blood sugar. Sicilian Rouge High GABA tomato (High GABA tomato) is the world's first tomato that has been approved as a gene-edited food, which uses CRISPR/Cas9 to enable a dense accumulation of GABA. In this presentation, we will report the background of the development of the GABA hyper-accumulating tomato as well as the results of an in-house monitoring survey conducted on the consumption of this tomato. Methods: Through gene editing technology, we have successfully modified the auto-inhibitor-domain of the glutamate decarboxylase gene to enhance its activity and high GABA tomatoes were produced. Thirteen in-house monitors (5 normotensive and 8 mildly hypertensive) were asked to consume the high GABA tomatoes starting in November 2021. High GABA tomatoes were consumed fresh or cooked, with a daily dosage of 5 tomatoes. The monitored participants had their home blood pressure measured twice a day, in the morning and at night, and their blood pressure was compared between the two weeks before and the two weeks after the start of tomato consumption. Results: High GABA tomatoes contain 95 mg/100 g of GABA, and eating 5 tomatoes per day provided approximately 100 mg of GABA. No clinically significant blood pressure changes were observed in the five participants with average baseline home systolic blood pressure < 135 mmHg. When the eight participants with baseline systolic blood pressure > = 135 mmHg were divided into groups, the systolic blood pressure in 3 out of 4 subjects in the upper half decreased significantly (from 155 mmHg to 150 mmHg, Δ-4.6 mmHg). On the other hand, systolic blood pressure of the lower half of the hypertensives increased significantly in 2 subjects, but the mean did not change significantly (from 137 mmHg to 139 mmHg, Δ + 1.8 mmHg). Conclusion: While further study is necessary, the results suggest that Sicilian Rouge High GABA tomato may contribute to blood pressure reduction when consumed by hypertensive individuals.

Human iPSC-derived neural stem cells with ALDH5A1 mutation as a model of succinic semialdehyde dehydrogenase deficiency

BackgroundSuccinic semialdehyde dehydrogenase deficiency (SSADH-D) is an autosomal recessive gamma-aminobutyric acid (GABA) metabolism disorder that can arise due to ALDH5A1 mutations, resulting in severe, progressive, untreatable neurodegeneration. SSADH-D is primarily studied using simplified models, such as HEK293 cells overexpressing genes of interest, but such overexpression can result in protein aggregation or pathway saturation that may not be representative of actual underlying disease phenotypes.MethodsWe used a CRISPR/Cas9 approach to generate human iPSC cell lines bearing ALDH5A1 mutations. Through screening, two different mutant cell lines, NM_001080.3: c.727_735del (p.L243_S245del) and NM_001080.3: c.730_738del (p.A244_Q246del), were obtained. We induced iPSCs to neural stem cells and analyzed the characteristics of ALDH5A1 mutations in stem cells.ResultsThe human iPSC and NSC cell lines presented typical stem cell–like morphology. We found changes in ALDH5A1 expression and GABA accumulation in the different cell lines. In addition, by analyzing the cDNA between the wild-type and the mutant cell lines, we found that the mutant cell lines had a splicing variant.ConclusionsiPSCs represent a promising in vitro model for SSADH-D that can be used to study early central nervous system developmental alterations and pathogenic mechanisms.

A novel γ-aminobutyric acid biosynthetic pathway in Lentilactobacillus curieae CCTCC M 2011381T

Gamma-aminobutyric acid (GABA) is an important neurotransmitter for mammals, thus biosynthesized or bio-transformed GABA is an important compound for functional foods. In this study, unique biosynthetic pathways of GABA in a novel lactic acid bacteria (LAB) strain, Lentilactobacillus curieae CCTCC M 2011381T, was discovered. L-glutamate decarboxylase (GAD) which generally catalyzes L-glutamate decarboxylation to form GABA in bacteria, is absent in this strain. Alternatively, transcriptional analysis revealed that L-glutamate played as an amino donor for the step through which succinic semialdehyde (SSA) was converted to GABA catalyzed by GABA transaminase (GABA-T). Then, the deletion and complementation of the GABA-T gene confirmed that GABA-T was the key enzyme responsible for GABA accumulation from SSA. According to residual GABA titer could be detected after the deletion of the GABA-T gene, other decarboxylases among the genome were analyzed by RT-PCR. 5-Oxopent-3-ene-1,2,5-tricarboxylate decarboxylase (HpaG) was verified possessing weaker catalytic capability for the decarboxylation of L-glutamate based on the hetero-expression of the enzyme in E. coli. Thus, GABA is produced mainly through the transamination reaction on SSA catalyzed by GABA-T and L-glutamate as the amino donor, and slightly from the decarboxylation of L-glutamate catalyzed by HpaG in L. curieae, which are distinct pathways in LAB.

Differential Regulation of Amino Acids and Nitrogen for Drought Tolerance and Poststress Recovery in Creeping Bentgrass

Effects of amino acids and nitrogen on plant regrowth or recovery from drought stress remain largely unknown. The objectives of this study were to examine how gamma-aminobutyric acid (GABA) or proline, alone and in combination, or inorganic nitrogen [ammonium nitrate (NN)] may differentially affect turf performance during drought stress and rewatering, and to determine which specific endogenous amino acids regulated by GABA, proline, or NN priming were associated with plant tolerance to drought stress and postdrought recuperation in cool-season grass species. Creeping bentgrass (Agrostis stolonifera cv. Penncross) planted in porous ceramic fritted clay medium were exposed to well-watered conditions or drought stress by withholding irrigation for 21 days in growth chambers. Plants were treated with water (untreated control), GABA, or proline alone and in combination, or NN through foliar spray before drought stress and every 7 days during the 21-day stress period. For postdrought recovery, at 21 days of drought treatment, plants were rewatered for 14 days to return soil water content to prestress levels. Plants treated with GABA or proline alone or in combination maintained higher turf quality (TQ), dark green color index (DGCI), and stolon length by 21 days of drought stress, whereas proline-treated plants also maintained higher leaf relative water content (RWC) during drought as well as longer stolon length during rewatering. Plants treated with NN maintained higher TQ and leaf RWC during drought and had improved percent canopy cover, DGCI, and stolon length during postdrought rewatering. Accumulation of endogenous amino acids under drought stress, including proline and alanine, for proline-treated creeping bentgrass may have contributed to the enhancement of drought tolerance and postdrought regrowth. Nitrogen-enhanced accumulation of GABA, proline, and glutamic acid may have played a role in active amino acid assimilation and subsequent postdrought regrowth. Results from this study indicate that GABA or proline were mainly effective in promoting the tolerance of creeping bentgrass to drought stress while inorganic NN was effective in promoting rapid postdrought recovery and regrowth potential through the activation of amino acid metabolism. Endogenous amino acids, including GABA, proline, alanine, and glutamic acid, may be used as biomarkers to select for drought-tolerant plants and biostimulant components for improvement of drought stress tolerance and poststress recovery in cool-season turfgrass species.

Fast-acidification promotes GABA synthesis in response to acid stress in Streptococcus thermophilus

Most Streptococcus thermophilus strains possess the conserved gadBC gene cluster for gamma-aminobutyric acid (GABA) synthesis, endowing yogurt with special beneficial characteristics. Here, we found that GABA yields of S. thermophilus were always positively correlated with fast-acidification. To explore the regulation mechanism of acid production to GABA synthesis, the activities of β-galactosidase (LacZ), lactate dehydrogenase (Ldh) and glutamate decarboxylase (GAD) were determined in strains SDMCC050243 and SDMCC050242. The results showed the higher enzyme activities of LacZ and Ldh at the exponential growth phases, the higher GAD activity. Furthermore, the activity of PgadB promoter responding to the transcription of gadB gene increased with the decrease of pH, suggesting that acid production activated the expression of GAD to synthesize GABA. Meanwhile, the viability of S. thermophilus increased with the accumulation of GABA, confirming the protection role of GABA synthesis against acid stress. In milk fermentation using ten GABA-producing strains, the more GABA yields were always accompanied with the shorter curd time, further illustrating that fast-acidification was beneficial to GABA synthesis. Therefore, this study revealed that fast-acidification promoted GABA biosynthesis in S. thermophilus, subsequently protected strains against acid stress, providing theoretical foundations for making the GABA-enriched yogurt.

Exogenous methyl jasmonate enhances phytochemicals and delays senescence in harvested strawberries by modulating GABA shunt pathway

In order to illustrate the relationship between methyl jasmonate (MeJA) and gamma aminobutyric acid (GABA) in maintaining the quality and improving the postharvest life of strawberry fruit, the harvested fruit were treated with MeJA for 16 h at 20 °C and stored at 3 ± 0.5 °C for 12 days. MeJA enhanced the expression levels of GABA shunt pathway-related genes, including glutamate decarboxylase, GABA transaminase, and succinic semialdehyde dehydrogenase, leading to an increase in GABA accumulation. Treated fruit showed higher levels of total acids, anthocyanins, total phenolics, antioxidants, and phenylalanine ammonia-lyase activity, and lower levels of cell membrane deterioration, total soluble solids, polyphenol oxidase activity and decay incidence rate. The results suggest that the positive effects of MeJA in extending the fruit postharvest life, enhancing phytochemical compounds, and decreasing the decay incidence rate may be due to the effects on motivating GABA shunt pathway and PAL enzyme activity.

Ultrasound accelerated γ-aminobutyric acid accumulation in coffee leaves through influencing the microstructure, enzyme activity, and metabolites

Gamma-aminobutyric acid (GABA) is a non-protein amino acid that possesses various physiological functions. Our previous study has shown that ultrasound increased GABA accumulation in coffee leaves. In this study, we aimed to uncover the GABA enrichment mechanism by investigating the surface microstructure, cellular permeability, enzyme activities, and metabolomics of coffee leaves under ultrasound treatment. The results showed that ultrasound increased the electrical conductivity and the activities of glutamate decarboxylase, γ-aminoaldehyde dehydrogenase, and diamine oxidase by 12.0%, 265.9%, 124.1%, 46.8%, respectively. Environmental scanning electron microscope analysis demonstrated an increased opening of stomata and the rougher surface in the leaves after ultrasound treatment. UPLC-qTOF-MS/MS-based untargeted metabolomics analysis identified 82 differential metabolites involved in various metabolism pathways. Our results indicated that ultrasound changed the surface microstructure of coffee leaves, thereby accelerating the migration of glutamate into the cells; activated related enzymes; regulated C/N metabolism pathways, which led to an increase of GABA.

EP013: Novel biomarkers of succinic semialdehyde dehydrogenase deficiency highlight opportunities for screening and detection of GABA catabolism pathway abnormalities

GABA (gamma-aminobutyric acid) is catabolized in the mitochondrial matrix via the GABA shunt, a 2-step enzymatic pathway which encompasses the transamination of GABA to succinic semialdehyde by GABA-transaminase (GABA-T), followed by oxidation to succinate by succinic semialdehyde dehydrogenase (SSADH). Deficiency of SSADH results in accumulation of GABA and reduction of succinic semialdehyde to gamma-hydroxybutyrate (GHB), the principal neurotoxin in SSADH deficiency (SSADHD). SSADHD is an autosomal recessive disorder associated with biallelic pathogenic variants in ALDH5A1, typically characterized by developmental delay, hypotonia, intellectual disability, ataxia, seizures, hyperkinetic behavior, aggression, psychiatric disorders, and sleep disturbances.

Ultrasonication increases γ‐aminobutyric acid accumulation in coffee leaves and affects total phenolic content and angiotensin‐converting enzyme inhibitory activity

In this study, the accumulation of γ-aminobutyric acid (GABA) in coffee leaves under ultrasonication and the impacts on total phenolic content (TPC), the activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), and angiotensin-converting enzyme (ACE) were investigated. The pH, incubation time, and ultrasonic power were the three most important factors that affected the accumulation of GABA. Coffee leaves treated with 20/40 kHz ultrasound have the highest GABA and the greatest GAD and ACE inhibitory activity. After optimization with a Box–Behnken design, GABA content was increased by 1.6 times under the optimal conditions of pH 5.9, incubation time 6.6 hr, and ultrasonic power 150 W. Our study indicated that ultrasonication activated GAD while inhibited GABA-T, thereby leading to an increase in GABA and ACE inhibitory activity. Ultrasonication also enhanced TPC; however, long time incubation caused the oxidation of phenolic compounds, and thus, affected the ACE inhibitory activity. Novelty impact statement This is the first research that studied the enrichment of Gamma-aminobutyric acid (GABA) in coffee leaves using ultrasound stress. GABA-enriched coffee leaves can be used to prepare tea-like beverage or natural health products to prevent hypertension, anxiety, and sleeplessness.

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.

Screening and characterisation of gamma-aminobutyric acid (GABA) producing lactic acid bacteria isolated from Thai fermented fish (Plaa-som) in Nong Khai and its application in Thai fermented vegetables (Som-pak)

This study aimed to evaluate the capability of lactic acid bacteria (LAB) isolated from different kinds of fermented fish products (Plaa-som) on the production of γ-aminobutyric acid (GABA). Among them, isolate L10-11, identified as Lactobacillus plantarum, offered the highest GABA production and was selected for further study. The highest production of GABA was obtained within 48h from the de Man, Rogosa and Sharpe (MRS) medium, having a pH range of 5-6. Increasing the monosodium glutamate (MSG) concentration resulted in a higher accumulation of GABA and reached the highest concentration (15.74 g/L) using 4% (w/v) MSG, while high residual MSG was also observed in accordance with concentration increase. On the other hand, the addition of NaCl in the culture medium by up to 7% (w/v) did not suppress GABA production. Preliminary application of strain L10-11 as starter producing GABA was investigated in Thai fermented vegetables (Som-pak). It was found that GABA formation could be observed increasingly following the concentration of MSG added. GABA content in Som-pak was 5 times higher than that of control when inoculating Lb. plantarum L10-11 and adding MSG at 1% (w/v). In addition, sensory evaluation revealed that addition of this starter culture to Som-pak gave overall acceptability slightly higher than that which allowed fermentation to occur spontaneously. This alternative procedure would be successful for improving the nutritional quality of functional fermented food.

Effect of sucrose on antioxidant activities and other health-related micronutrients in gamma-aminobutyric acid (GABA)-enriched sprouting Southern Vietnam brown rice

Improving the nutritional quality of germinated brown rice (GBR) is important for the population consuming rice as part of the daily diet to offer health benefits. In this study, a set of popularly grown 79 rice varieties in Southern Vietnam was evaluated to identify the potential materials for producing gamma-aminobutyric acid (GABA)-enriched GBR. Enhancing antioxidant components, antioxidant activities, and other health-promoting micronutrients by sucrose treatment were also investigated in both white and red GBR. The results indicated that OM457 (white rice) and TN11 (red rice)-derived GBR had the highest GABA content of 105.68 mg GABA/100 g F.W and 47.68 mg GABA/100 g F.W among investigated rice varieties, respectively. Sucrose at 50 mM, in general, boosted total phenolic compounds, total flavonoids, anthocyanin content, thereby increasing antioxidant activities in both these GBR. Moreover, GC-MS-based metabolomic analysis indicated that GABA accumulation under sucrose treatment was rice pericarp/variety-specific. Also, sucrose stimulated the accumulation of other health-benefiting metabolites and free sugars. Altogether, the results in this study indicate that sucrose is an effective elicitor for improving the nutritional value in GBR, facilitating for production of ready-to-eat GBR with health-promoting benefits.

Characteristics of Potential Gamma-Aminobutyric Acid-Producing Bacteria Isolated from Korean and Vietnamese Fermented Fish Products.

Gamma-aminobutyric acid (GABA) is a neurotransmitter that exerts several physiological functions and positive effects on human health. The aim of this study was to isolate and characterize the strains that had GABA-producing abilities from various fermented fish products. A total of 91 acid-producing strains were isolated from 41 samples of fermented fish products, and 27 strains showing GABA-producing abilities were identified by the 16S rDNA sequences. Among the strains, 31% strains tolerated at high-salt environment of 10-20% throughout the fermentation of fish sauces. The 27 isolates that produced GABA at various concentrations did so in the range of 5 to 454 mM. These GABA-producing isolates were identified as lactic acid bacteria of 14 strains, which included twelve Lactococcus lactis, one Enterococcus faecium, and one Lactococcus pentosus; eight Bacillus cereus group, which included seven B. thuringiensis and one B. cereus; and five Staphylococcus spp. Interestingly, with Vietnamese fish sauces, we mostly identified species of B. thuringiensis and Staphylococcus spp., while with Korean fermented fish products, the majority of the strains identified belonged to L. lactis. Among the strains, B. thuringiensis LH2134 produced the highest levels of GABA at 366 mM among the strains identified from Vietnamese fish sauces, whereas L. lactis LA43, a new strain isolated from Korean jeotgal (salted shrimp paste), produced the highest amount of GABA at 454 mM and the glutamate concentration in the medium was essential for GABA accumulation. Therefore, such the isolates might serve as good starters for development of more GABA-reinforced foods among fermented fish products.

Biosynthesis and accumulation of GABA in rice plants treated with acetic acid.

Rice seedlings (Oryza sativa) that have died from drought cannot be rescued by watering afterward, but pre-treatment with exogenous acetic acid enabled the plants to produce shoots again after being watered (hereinafter referred to as "drought resilience"). To elucidate the metabolism of acetic acid, we treated rice plants with 13C-labeled acetic acid and traced 13C-labeled metabolites using LC-MS and 13C-NMR techniques. The LC-MS and 13C-NMR spectral data of the root extracts indicated that the acetic acid treatment was absorbed into the plants and then was metabolized to gamma-aminobutyric acid (GABA) by glutamic acid decarboxylase (GAD). GABA accumulation in the roots took place in advance of that in the shoots, and the survival rate against drought stress increased in proportion to the amount of GABA accumulated in the shoots. Therefore, GABA accumulation in shoots may be a key step in drought resilience induced by the acetic acid treatment.

Enhanced accumulation of gamma-aminobutyric acid in rice bran using anaerobic incubation with various additives

An anaerobic incubation for the enzymatic production of gamma-aminobutyric acid (GABA) in rice bran with the addition of glutamate, hydrolyzed wheat protein (HWP), yeast extract (YE) and pyridoxal-5-phosphate (PLP) was investigated. Rice bran was moistened (30% moisture content) with an electrolyzed oxidizing water and anaerobically incubated under nitrogen at 40 °C for 8 h. The incubation activated the glutamate decarboxylase (GAD) in rice bran and increased the GABA content from 10.7 to 171.5 mg/100 g. The addition of glutamate and protein hydrolysates further amplified the GABA content in the treated rice bran: 974.9, 487.4, and 372.8 mg/100 g, with 2.25% glutamate, 6% HWP, and 8% YE, respectively. Furthermore, addition of PLP (1.48 mg/100 g) as a coenzyme for GAD, together with 2.25% glutamate addition, could raise the GABA accumulation in rice bran to 2242 mg/100 g. Other amino acids in rice bran were changed in their composition by the anaerobic treatment.

Enhanced productivity of gamma-amino butyric acid by cascade modifications of a whole-cell biocatalyst.

We previously developed a gamma-amino butyric acid (GABA)-producing strain of Escherichia coli, leading to production of 614.15g/L GABA at 45°C from L-glutamic acid (L-Glu) with a productivity of 40.94g/L/h by three successive whole-cell conversion cycles. However, the increase in pH caused by the accumulation of GABA resulted in inactivation of the biocatalyst and consequently led to relatively lower productivity. In this study, by overcoming the major problem associated with the increase in pH during the production process, a more efficient biocatalyst was obtained through cascade modifications of the previously reported E. coli strain. First, we introduced four amino acid mutations to the codon-optimized GadB protein from Lactococcus lactis to shift its decarboxylation activity toward a neutral pH, resulting in 306.65g/L of GABA with 99.14mol% conversion yield and 69.8% increase in GABA productivity. Second, we promoted transportation of L-Glu and GABA by removing the genomic region encoding the C-plug of GadC (a glutamate/GABA antiporter) to allow its transport path to remain open at a neutral pH, which improved the GABA productivity by 16.8% with 99.3mol% conversion of 3ML-Glu. Third, we enhanced the expression of soluble GadB by introducing the GroESL molecular chaperones, leading to 20.2% improvement in GABA productivity, with 307.40g/L of GABA and a 61.48g/L/h productivity obtained in one cycle. Finally, we inhibited the degradation of GABA by inactivation of gadA and gadB from the E. coli genome, which resulted in almost no GABA degradation after 40h. After the cascade system modifications, the engineered recombinant E. coli strain achieved a 44.04g/L/h productivity with a 99.6mol% conversion of 3ML-Glu in a 5-L bioreactor, about twofold increase in productivity compared to the starting strain. This increase represents the highest GABA productivity by whole-cell bioconversion using L-Glu as a substrate in one cycle observed to date, even better than the productivity obtained from the three successive conversion cycles.