Gamma-aminobutyric Acid Production Research Articles

Microbial chassis design and engineering for production of gamma-aminobutyric acid.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid which is widely applied in agriculture and pharmaceutical additive industries. GABA is synthesized from glutamate through irreversible α-decarboxylation by glutamate decarboxylase. Recently, microbial synthesis has become an inevitable trend to produce GABA due to its sustainable characteristics. Therefore, reasonable microbial platform design and metabolic engineering strategies for improving production of GABA are arousing a considerable attraction. The strategies concentrate on microbial platform optimization, fermentation process optimization, rational metabolic engineering as key metabolic pathway modification, promoter optimization, site-directed mutagenesis, modular transporter engineering, and dynamic switch systems application. In this review, the microbial producers for GABA were summarized, including lactic acid bacteria, Corynebacterium glutamicum, and Escherichia coli, as well as the efficient strategies for optimizing them to improve the production of GABA.

Specific transcription factors Ascl1 and Lhx6 attenuate diabetic neuropathic pain by modulating spinal neuroinflammation and microglial activation in mice

Gamma-aminobutyric acid (GABA) neuronal system-related transcription factors (TFs) play a critical role in GABA production, and GABA modulates diabetic neuropathic pain (DNP). The present study investigated the therapeutic effects of intrathecal delivery of two TFs achaete-scute homolog 1 (Ascl1) and LIM homeobox protein 6 (Lhx6) in a mouse model of DNP and elucidated their underlying mechanisms. GABA-related specific TFs, including Ascl1, Lhx6, distal-less homeobox 1, distal-less homeobox 5, the Nkx2.1 homeobox gene, and the Nkx2.2 homeobox gene, were investigated under normal and diabetic conditions. Among these, the expression of Ascl1 and Lhx6 was significantly downregulated in mice with diabetes. Therefore, a single intrathecal injection of combined lenti-Ascl1/Lhx6 was performed. Intrathecal delivery of lenti-Ascl1/Lhx6 significantly relieved mechanical allodynia and heat hyperalgesia in mice with DNP. Ascl1/Lhx6 delivery also reduced microglial activation, decreased the levels of pro-inflammatory cytokines including tumor necrosis factor-α and interleukin (IL)-1β, increased the levels of anti-inflammatory cytokines including IL-4, IL-10, and IL-13, and reduced the activation of p38, c-Jun N-terminal kinase, and NF-κB in the spinal cord of mice with DNP, thereby reducing DNP. The results of this study suggest that intrathecal Ascl1/Lhx6 delivery attenuates DNP via upregulating spinal GABA neuronal function and inducing anti-inflammatory effects.

Metabolic engineering of Corynebacterium glutamicum CGY-PG-304 for promoting gamma-aminobutyric acid production

Metabolic engineering of Corynebacterium glutamicum CGY-PG-304 for promoting gamma-aminobutyric acid production

Phenotypic, Technological, Safety, and Genomic Profiles of Gamma-Aminobutyric Acid-Producing Lactococcus lactis and Streptococcus thermophilus Strains Isolated from Cow's Milk.

Gamma-aminobutyric acid (GABA)-producing lactic acid bacteria (LAB) can be used as starters in the development of GABA-enriched functional fermented foods. In this work, four GABA-producing strains each of Lactococcus lactis and Streptococcus thermophilus species were isolated from cow's milk, and their phenotypic, technological, and safety profiles determined. Genome analysis provided genetic support for the majority of the analyzed traits, namely, GABA production, growth in milk, and the absence of genes of concern. The operon harboring the glutamate decarboxylase gene (gadB) was chromosomally encoded in all strains and showed the same gene content and gene order as those reported, respectively, for L. lactis and S. thermophilus. In the latter species, the operon was flanked (as in most strains of this species) by complete or truncated copies of insertion sequences (IS), suggesting recent acquisition through horizontal gene transfer. The genomes of three L. lactis and two S. thermophilus strains showed a gene encoding a caseinolytic proteinase (PrtP in L. lactis and PrtS in S. thermophilus). Of these, all but one grew in milk, forming a coagulum of good appearance and an appealing acidic flavor and taste. They also produced GABA in milk supplemented with monosodium glutamate. Two L. lactis strains were identified as belonging to the biovar. diacetylactis, utilized citrate from milk, and produced significant amounts of acetoin. None of the strains showed any noticeable antibiotic resistance, nor did their genomes harbor transferable antibiotic resistance genes or genes involved in toxicity, virulence, or pathogenicity. Altogether these results suggest that all eight strains may be considered candidates for use as starters or components of mixed LAB cultures for the manufacture of GABA-enriched fermented dairy products.

Optimization of fermentation for gamma-aminobutyric acid (GABA) production by Lactiplantibacillus plantarum Lp3 and the development of fermented soymilk

Gamma-aminobutyric acid (GABA) yield was taken as the index to optimize the fermentation medium and conditions for Lactiplantibacillus plantarum Lp3, and then fermented soymilk with high GABA yield by L. plantarum Lp3 was developed. GABA is a non-protein amino acid with multiple physiological functions. In recent years, L. plantarum strains have been involved in the metabolism of GABA and can serve as microbials for producing GABA. In this study, the medium and fermentation conditions for the production of GABA by L. plantarum Lp3 were optimized using response surface methodology (RSM). The results showed that the optimal concentrations of glucose, yeast extract, and Ca2+ were 20.20, 15.80, and 9.90 mg/mL, respectively, sodium glutamate was 5.00 mg/mL, fermentation temperature was 35 °C, initial pH was 6.4, and fermentation time was 46 h. Under the optimized conditions, the highest GABA yield was 3.74 mg/mL. In addition, the optimal fermentation conditions of fermented soymilk with high GABA yield by L. plantarum Lp3 were: L. plantarum Lp3 and Streptococcus thermophilus 2:1, sodium glutamate 0.4%, fermentation time 10 h, fermentation temperature 35 °C, and the final GABA yield was 0.552 mg/mL.

Intracellular Flux Prediction of Recombinant Escherichia coli Producing Gamma-aminobutyric Acid.

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

Genetic Circuits for Feedback Control of Gamma-Aminobutyric Acid Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Engineered microorganisms such as the probiotic strain Escherichia coli Nissle 1917 (EcN) offer a strategy to sense and modulate the concentration of metabolites or therapeutics in the gastrointestinal tract. Here, we present an approach to regulate the production of the depression-associated metabolite gamma-aminobutyric acid (GABA) in EcN using genetic circuits that implement negative feedback. We engineered EcN to produce GABA by overexpressing glutamate decarboxylase and applied an intracellular GABA biosensor to identify growth conditions that improve GABA biosynthesis. We next employed characterized genetically encoded NOT gates to construct genetic circuits with layered feedback to control the rate of GABA biosynthesis and the concentration of GABA produced. Looking ahead, this approach may be utilized to design feedback control of microbial metabolite biosynthesis to achieve designable smart microbes that act as living therapeutics.

Gamma Amino Butyric Acid (GABA) and Ferulic Acid Esterase (FAE) Producing Psychobiotic Bacteria Isolated from Cereal-Based Fermented Food.

Gut microbiota plays an important role in regulating enteric, immune and neural pathways. Many neuropsychiatric disorders such as anxiety, depression, autism and cognitive behaviour are associated with gut dysbiosis. Gamma-aminobutyric acid (GABA) and short-chain fatty acids produced by gut bacteria influence gastrointestinal and neurological functions. Ferulic acid esterase (FAE) which releases ferulic acid (FA) from feruloylated sugar ester conjugates, naturally found in grains, fruits and vegetables, is also produced by some gut bacteria and helps prevent neurodegeneration. These properties provide bacteria with the ability to maintain intestinal barrier function and prevent neuropsychiatric disorders. Therefore, this study aims to isolate GABA and FAE-producing LAB and characterize their bioactive and probiotic properties. A total of twelve cultures were isolated, of which eight bacteria positive for GABA, FAE and SCFA production were selected for further investigation. All selected bacteria were positive for bile salt hydrolase (BSH) and showed acid tolerance, resistance to bile salt, stimulated gastric and pancreatic juice, and auto- and co-aggregation properties. Furthermore, selected LAB showed mucin adhesion efficiency greater than 80% and exhibited γ-hemolytic activity. 16S rRNA sequencing identified NS0969, B1, C1, C2, M1, M2, and R2 as Limosilactobacillus fermentum and R1 as Lactiplantibacillus pentosus. This study showed that selected bacteria and/or their postbiotic preparations can be used as potential psychobiotics.

Antioxidant activity, GABA (γ-aminobutyric acid) production, and potential for colonization of Lactobacillus fermentum InaCC B1295 encapsulated with cellulose microfiber hydrogel from oil palm solid waste during storage

Lactobacillus fermentum InaCC B1295 is a probiotic bacterium that improves intestinal and immune systems, positively impacting human health. It produces various bioactive compounds, including bacteriocin, antioxidant enzymes, and gamma-aminobutyric acid (GABA), and colonizes the human gastrointestinal system. This study aimed to evaluate the antioxidant activity (IC50), GABA production, and colonization potential of L. fermentum InaCC B1295 with encapsulation by cellulose microfiber from oil palm solid waste (trunk, frond, leaf, and empty bunches) at room and refrigeration temperature with storage time 0, 14, and 28 days. The strain InaCC B1295 showed a high activity to scavenging ability against DPPH (1,1-diphenyl-2-picrylhydrazyl) for the treatment with encapsulation with oil palm empty bunches at refrigeration temperature after storage 14 days, which reached 88.56 ppm. Furthermore, L. fermentum InaCC B1295 produced GABA ranging from 0.435 mg/L to 2.215 mg/L. Lactobacillus fermentum InaCC B1295 encapsulated with oil palm leaf produced the most GABA at 0-day storage with a concentration of 2.215 mg/L and decreased during storage for all treatments. L. fermentum InaCC B1295 cells encapsulated in CMF hydrogel from empty oil palm fruit bunches had a lower autoaggregation value than free cells. The cells encapsulated with CMF hydrogel from oil palm empty bunches, on the other hand, had higher coaggregation and hydrophilicity values than the free cells (unencapsulated cells).

Biosynthesis of Gamma-Aminobutyric Acid (GABA) by Lactiplantibacillus plantarum in Fermented Food Production.

In recent decades, given the important role of gamma-aminobutyric acid (GABA) in human health, scientists have paid great attention to the enrichment of this chemical compound in food using various methods, including microbial fermentation. Moreover, GABA or GABA-rich products have been successfully commercialized as food additives or functional dietary supplements. Several microorganisms can produce GABA, including bacteria, fungi, and yeasts. Among GABA-producing microorganisms, lactic acid bacteria (LAB) are commonly used in the production of many fermented foods. Lactiplantibacillus plantarum (formerly Lactobacillus plantarum) is a LAB species that has a long history of natural occurrence and safe use in a wide variety of fermented foods and beverages. Within this species, some strains possess not only good pro-technological properties but also the ability to produce various bioactive compounds, including GABA. The present review aims, after a preliminary excursus on the function and biosynthesis of GABA, to provide an overview of the current uses of microorganisms and, in particular, of L. plantarum in the production of GABA, with a detailed focus on fermented foods. The results of the studies reported in this review highlight that the selection of new probiotic strains of L. plantarum with the ability to synthesize GABA may offer concrete opportunities for the design of new functional foods.

Complete genome sequence of the probiotic Bifidobacterium adolescentis strain iVS-1

ABSTRACT Bifidobacterium adolescentis iVS-1 is a human-isolated strain known to possess several probiotic properties. Here, its genome was completely sequenced to examine genes associated with lactose metabolism and other potentially beneficial traits, such as the production of folate and gamma-aminobutyric acid (GABA).

Spent coffee grounds as a potential culture medium for γ-aminobutyric acid (GABA) production by Levilactobacillus brevis PML1

This study aimed to investigate the Gamma-aminobutyric acid (GABA) production potential of Levilactobacillus brevis PML1 using an industrial waste-based culture medium. The optimization of GABA production was carried out in Spent Coffee Grounds (SCG) medium employing Response Surface Methodology (RSM). A Central Composite Design (CCD) was employed, revealing that the optimal SCG treatment conditions were as follows: 6% H2SO4 concentration, 120 min incubation, 0.14 mL Accellerase-1000 enzyme. High-performance chromatography analysis demonstrated a GABA yield of 492 mg/L after 48 h at 37 °C when supplemented with 3% monosodium glutamate. The antimicrobial activity assessment exhibited sensitivity towards Staphylococcus aureus among tested pathogenic bacteria. Scanning electron microscopy elucidated the antibacterial mechanism on Escherichia coli and S. aureus as representative strains. Furthermore, radical inhibitory activities were observed through DPPH assay recording a maximum inhibition rate of 13.15%, while FRAP assay indicated an antioxidant capacity of up to 0.419 mmol/L. Also, L. brevis demonstrated 53.40% cholesterol removal capability. Cytotoxic effects on human colon cancer cell lines Caco-2 were examined using MTT assay, yielding an IC50 value of approximately 3.25 g/mL for the extracts. In conclusion, this study highlights that SCG can serve as an affordable culture medium for efficient metabolite production.

Gamma-Aminobutyric Acid (GABA) Producing Lactobacillus plantarum TSUB-17 and Probiotic Properties for Using as Probiotics Additive in Swine Feed

Due to the use of probiotics in animal husbandry has recently increased, the selection of high-quality probiotic strains is crucial. Since the uses of probiotics for animal feed supplements have to be applied at high doses of bacteria to be effective, the cultures’ safety is essential. This study successfully isolated the novel Lactobacillus sp. from swine fecal samples and identified it as L. plantarum TSUB-17. The L. plantarum TSUB-17 exhibited excellent probiotic properties, including 100 % resistance to 3 % acids and 1 % bile salts, and effectively inhibits pathogenic bacteria such as Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Salmonella Typhimurium (isolated ESBL strain). L. plantarum TSUB-17 was also found to adhere to the intestinal wall and form a biofilm. Furthermore, the bacteria have revealed some unique properties of GABA production and detected the glutamate decarboxylase B (gadB) gene similar to L. brevis both in nucleotide sequence and secondary structure of the glutamate decarboxylase enzyme that was deduced from the nucleotide. The isolated bacteria also have antioxidant activity, and most importantly, the isolated strain was safe; it is non-hemolysis and susceptible to the tested antimicrobials. Therefore, L. plantarum TSUB-17 is suitable for further development as a probiotic additive in animal feed.  HIGHLIGHTS This study successfully isolated Lactobacillus plantarum TSUB-17 with a high potential to develop as a probiotic for animals. The isolated bacterium has excellent properties: It tolerates high levels of acids and bile salts. It is effective against many pathogenic bacteria, produces gamma-aminobutyric acid (GABA) and shows high levels of antioxidant activity. Moreover, L. plantarum TSUB-17 is very safe due to its non-hemolytic strain and susceptibility to numerous antimicrobial drugs. GRAPHICAL ABSTRACT

Exploring autochthonous strains with probiotic potential: A comprehensive characterization of functional properties and their application in fermented blueberry-watermelon smoothies

Fruit-fermented smoothies enriched with probiotics present a promising alternative for individuals who avoid dairy products. However, identifying suitable probiotic strains poses a critical challenge. This study aimed to characterize five potential probiotic strains: Hanseniaspora opuntiae BIOTEC045, Pediococcus pentosaceus BIOTEC046 and BIOTEC047, and Lactiplantibacillus plantarum LpAv and 73a. The objective was to assess their aggregation and antipathogenic abilities, safety, stress tolerance, and other functional properties. LpAv and 73a displayed the most favorable characteristics. They exhibited high auto-aggregation (>65%), resistance to simulated gastrointestinal conditions (with only 0.3–1.5 log reductions), efficient enzyme production (high levels of β-galactosidase and β-glucosidase), capability to utilize various prebiotics (with maximum optical density values of 1.62–1.81), and production of gamma-aminobutyric acid (1.2 mM), among other features. LpAv and 73a were selected for fermenting blueberry-watermelon smoothies (BWS). During a 7-day storage period at 5 °C, both strains maintained high viability (>9 log colony forming units/mL), while the control samples exhibited spoilage microbial load, highlighting the effectiveness of lactic acid fermentation as a viable bio-preservation technique. Additionally, the BWS retained bioactive compounds and antioxidant activity throughout storage after fermentation, with total phenolic and anthocyanin contents of 0.68–0.73 mg/g and 0.16–0.17 mg/g, respectively. These findings confirm the suitability of BWS as a matrix for fermentation with the selected potential probiotic bacteria. Additionally, the comprehensive characterization of the strains provides valuable insights into their functional properties and contributes to the understanding of their potential health benefits.

Enhanced Production of Gamma-Aminobutyric Acid (GABA) from Lactobacillus futsaii CS3 Using Agri-Food Industries By-Products Under Batch and Fed-Batch Fermentation.

Enhanced Production of Gamma-Aminobutyric Acid (GABA) from Lactobacillus futsaii CS3 Using Agri-Food Industries By-Products Under Batch and Fed-Batch Fermentation.

Anti-fatigue activities of γ-aminobutyric acid-enriched soymilk in an acute exercise-treated mouse model via regulating AMPK/PGC-1α pathway

Gamma-aminobutyric acid (GABA), a four carbon non-protein amino acid, is extensively distributed in nature and shows various biological functions. In the present study, Lactobacillus brevis ATCC 367 and Lactobacillus plantarum ASCC 276, two producers with high GABA production capacity, were selected to ferment soymilk and their optimal culture conditions were investigated. Then, the anti-fatigue ability of GABA-enriched soy protein isolate (SPI) fermented milk was assessed by the weight-loaded forced swimming test and the related biochemical parameters were recorded to find possible reasons. Results demonstrated that soymilk added with glucose was capable of yielding the highest GABA content when used Streptococcus thermophilus 1303 and Lactobacillus brevis ATCC 367 as starter bacteria, and exhibited the high anti-fatigue effect in mice, mainly manifested in extending the forced swimming time, suppressing the blood lactic acid, urea nitrogen and lactate dehydrogenase content, and promoting the level of liver glycogen, muscle glycogen and muscle ATP. More importantly, SPI fermented milk beverages decreased reactive oxygen species levels via improving the concentrations of superoxide dismutase and glutathione peroxidase and inhibiting the content of malondialdehyd and promoted energy reserves by stimulating the expression of AMPK and PGC-1α in muscle, thus showing its fatigue-alleviating effect. Our study disclosed that there was a positive correlation between GABA content and anti-fatigue-related indicators of SPI fermented milk, which will be of interest for developing fermented milk with high anti-fatigue activity.

Optimization of Gamma-Aminobutyric Acid Production by Lactiplantibacillus plantarum FRT7 from Chinese Paocai.

Gamma-aminobutyric acid (GABA) is a widely available non-protein amino acid whose physiological importance goes beyond its role as an inhibitory neurotransmitter in mammals. The GABA synthesis ability of ten strains of Lactiplantibacillus plantarum was screened. They produced GABA ranging from 48.19 ± 3.44 to 100.75 ± 1.63 mg/L at 24 h-cultivation. Among them, Lp. plantarum FRT7 showed the highest GABA production. Therefore, FRT7 was chosen for GABA yield optimization. A one-factor-at-a-time strategy analysis of the GABA yield of FRT7 was performed, including the culture temperature, incubation time, inoculum volume, initial pH, the initial amount of monosodium glutamate (MSG), and pyridoxal 5'-phosphate (PLP) concentration, based on which the response surface methodology (RSM) was performed. After being cultured in an MRS culture medium supplemented with 3% MSG and 2 mmol/L of PLP at 40 °C with an initial pH of 7.0 for 48 h, the GABA reached a maximum yield of 1158.6 ± 21.22 mg/L. The results showed the experimental value of the GABA yield was in good agreement with the predicted values. Furthermore, the results from the RSM also indicated that the initial MSG addition, PLP concentration, and incubation time were significant variables. These results suggest that Lp. plantarum FRT7 has the potential to be a health-beneficial probiotic with commercial capabilities.

Gamma-aminobutyric acid (GABA) promotes characteristics of Levilactobacillus sp. LB-2

γ-Aminobutyric acid (GABA) is a pharmaceutical, bioactive amino acid that can be produced by lactic acid bacteria (LAB), but the yield of GABA is still limited by several reasons. In this study, a GABA-producing LAB strain Levilactobacillus sp. LB-2 (OP890629) was isolated from a Chinese traditional dough fermentation starter of Jiaozi. When subjected to GABA production optimization applying one-factor-at-a-time (OFAT) and response surface method (RSM), the temperature, pH, and L-monosodium glutamate (L-MSG) concentration for GABA production were set as 37 °C, pH 4.5, and 2.5 g/L, respectively. The maximum amount of GABA produced by strain Levilactobacillus sp. LB-2 was 49.42 g/L, which was 3.5-fold higher than that before optimization of 14.19 g/L. The GABA content of steamed bread made by Levilactobacillus sp. LB-2 significantly increased. Thus, strain Levilactobacillus sp. LB-2 could be used for the production of GABA or applied for the development and improvement of GABA-enriched foods.

Production of GABA in milk fermented by Bifidobacterium adolescentis strains selected on the bases of their technological and gastrointestinal performance

There is an increasing interest in producing foods enriched in gamma-aminobutyric acid (GABA), due to their purported health promoting attributes. GABA is the main inhibitor neurotransmitter of the central nervous system, and several microbial species are capable to produce it through decarboxylation of glutamate. Among them, several lactic acid bacteria species have been previously investigated as an appealing alternative to produce GABA enriched foods via microbial fermentation. In this work we report for the first time an investigation into the possibility of utilizing high GABA-producing Bifidobacterium adolescentis strains as a mean to produce fermented probiotic milks naturally enriched in GABA. To this end, in silico and in vitro analyses were conducted in a collection of GABA-producing B. adolescentis strains, with the main goal to scrutinize their metabolic and safety traits, including antibiotic resistance patterns, as well as their technological robustness and performance to survive a simulated gastrointestinal passage. One of the strains, IPLA60004, exhibited better survival to lyophilization and cold storage (for up to 4 weeks at 4 °C), as well as survival to gastrointestinal passage, as compared to the other strains under investigation. Besides, the elaboration of milk drinks fermented with this strain, yielded products with the highest GABA concentration and viable bifidobacterial cell counts, achieving conversion rates of the precursor, monosodium glutamate (GMS), up to 70 %. To our knowledge, this is the first report on the elaboration of GABA enriched milks through fermentation with B. adolescentis.

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

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