Transcriptomics reveal strain-specific metabolic strategies for acid resistance and gamma-aminobutyric acid (GABA) production in Levilactobacillus brevis.
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ABSTRACT: Background: Of 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 functionhealth outcomes. Understanding the molecular characteristics of GABA production by microbes within the gut can offer insight to novel therapies for mental health. Results: Three 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) 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 14689 after the stationary phase. The global gene expression during GABA production was determined by RNA sequencing at several timepoints. The GAD operon, responsible for GABA production and secretion, activated in Lbr-6108 after only six hours 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. Conclusions: This study is the first to evaluate whole transcriptome changes in L. brevis during GABA production over multiple timepoints. 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.
Project description:Purpose: High γ-aminobutyric acid (GABA)-producing Levilactobacillus brevis strain NPS-QW 145 along with Streptococcus thermophilus (one of the two starter bacteria used to make yogurt for its proteolytic activity) to enhance GABA production in milk. But a mechanistic understanding on how Levilactobacillus brevis cooperated with S. thermophilus to stimulate GABA production has been lacking. Method: Metatranscriptomic analyses combined with peptidomics were carried out to unravel the casein and lactose utilization patterns during milk fermentation with the co-culture. Results: We found particular peptides hydrolyzed by S. thermophilus 1275 were transported and biodegraded with peptidase in Lb. brevis 145 to meet the growth needs of the latter. In addition, amino acid synthesis and metabolism in Lb. brevis 145 were also activated to further support its growth. Glucose, as a result of lactose hydrolysis by S. thermophilus 1275, but not available lactose in milk, was outcompeted by Lb. brevis 145 as a main carbon source for glycolysis to produce ATP.In the stationary phase, under the acidic condition due to accumulation of lactic acid produced by S. thermophilus 1275, genes expression involved in pyridoxal phosphate (coenzyme of glutamic acid decarboxylase) metabolism and glutamic acid decarboxylase (Gad) in Lb. brevis 145 were induced for GABA production.
Project description:Gamma-aminobutyric acid (GABA) is a non-protein amino acid widespread in Nature. Among the various uses of GABA, its lactam form 2-pyrrolidone can be chemically converted to the biodegradable plastic polyamide-4. In metabolism, GABA can be synthesized either by decarboxylation of L-glutamate or by a pathway that starts with the transamination of putrescine. Fermentative production of GABA from glucose by recombinant Corynebacterium glutamicum has been described via both routes. Putrescine-based GABA production was characterized by accumulation of by-products such as N-acetyl-putrescine. Their formation was abolished by deletion of the spermi(di)ne N-acetyl-transferase gene snaA. To improve provision of L-glutamate as precursor 2-oxoglutarate dehydrogenase activity was reduced by changing the translational start codon of the chromosomal gene for 2-oxoglutarate dehydrogenase subunit E1o to the less preferred TTG and by maintaining the inhibitory protein OdhI in its inhibitory formby changing amino acid residue 15 from threonine to alanine. Putrescine-based GABA production by the strains described here led to GABA titers up to 63.2 g L-1 in fed-batch cultivation at maximum volumetric productivities up to 1.34 g L-11 h1-1, the highest volumetric productivity for fermentative GABA production reported to date. Moreover, GABA production from the carbon sources xylose, glucosamine, and N-acetyl-glucosamine that do not have competing uses in the food or feed industries was established.
Project description:Bacillus methanolicus is the next workhorse in biotechnology using methanol, an alternative and economical one-carbon feedstock that can be obtained directly from carbon dioxide, as both carbon and energy source for the production of various value-added chemicals. The wild-type strain MGA3 of B. methanolicus naturally overproduces l-glutamate in methanol-based fed-batch fermentations. Here we generated, by directed evolution, a B. methanolicus mutant strain exhibiting enhanced l-glutamate production capability (> 150%). To showcase the potential of the evolved strain, further metabolic engineering enabled the production of γ-aminobutyric acid (GABA) directly from l-glutamate, with a yield of >13 g/L from methanol during fed-batch fermentations. By using a system level analysis, encompassing whole-genome sequencing, RNA sequencing, fluxome analysis and metabolic modelling, we were able to elucidate the metabolic and regulatory adaptations that sustain the biosynthesis of these products. The metabolism of the mutant strain evolved to prioritize energy conservation and efficient carbon utilization. Key metabolic shifts include the downregulation of energy-intensive processes such as flagellation and motility and the rerouting of carbon fluxes towards α-ketoglutarate and its derivative, l-glutamate. Moreover, we observed that transformation of the evolved strain with a GABA biosynthesis plasmid had a positive effect on l-glutamate production and explained it by an upregulation of various transaminases involved in the l-glutamate biosynthesis from α-ketoglutarate. These insights provide a foundation for further rational metabolic engineering and bioprocess optimization, enhancing the industrial viability of B. methanolicus for sustainable production of l-glutamate and its derivatives.
Project description:Transcriptomics reveal strain-specific metabolic strategies for acid resistance and gamma-aminobutyric acid (GABA) production in Levilactobacillus brevis.
Project description:Various types of cancers, including colorectal cancer, have shown an unusual dependence on glutamine. Glutamine can undergo an alternate fate in neurons: conversion into a non-proteinogenic amino acid gamma-aminobutyric acid (GABA) by the glutamic acid decarboxylase (GAD), including two family members GAD1 and GAD2. Some studies have shown that GAD1 expression is dysregulated in certain cancer types, but the effect of GAD1 on the tumorigenic process remain largely unknown. Thus, we use mouse colon adenocarcinoma MC38 tumor samples to perform RNA-sequencing (RNA-seq) assay. The goal of this study is to investigate GAD1-mediated transcriptome changes within tumors.
Project description:Corynebacterium glutamicum shows a great potential for the production of gamma-aminobutyric acid (GABA) from glucose fermentation via putrescine. GABA, a non-protein amino acid widespread in nature, is a component of pharmaceuticals, foods and the biodegradable plastic polyamide 4. Here, the effect of GABA in the growth of C. glutamicum was evaluated. It was estimated that the presence 1.1 M of GABA in the medium reduces the maximum growth rate of C. glutamicum to half. It was also shown that the presence of GABA in the medium negatively affects the growth of C. glutamicum in ethanol as sole carbon source. Furthermore, a new route for the production of GABA in C. glutamicum was established. GABA production from glucose fermentation via putrescine was achieved by plasmid-based overexpression of putrescine transaminase (PatA) and gamma-aminobutyraldehyde dehydrogenase (PatD) in a putrescine production strain. The resultant strain can produce 5.3 ± 0.1 g L-1 of GABA. GABA production was improved by avoiding the formation of N-acetylputrescine and by reducing the amount of nitrogen in CGXII medium. Deletion of the genes responsible for GABA catabolism and GABA re-uptake led to an increase in the GABA production of 21% achieving a titer 8.0 ± 0.3 g L-1 and an increase in the volumetric productivity of 41% reaching a productivity of 0.31 g L-1 h-1, the highest volumetric productivity achieved so far for GABA production in C. glutamicum from glucose fermentation in flasks fermentations. The results obtained hitherto are very promising and competitive compared to the traditional pathway for the production of GABA.
Project description:The rostral ventrolateral medulla (RVLM), a part of the medullary reticular formation, plays a major role in several physiological responses, including cardiovascular and sympathetic nervous system functions. Although aging causes disturbances in the responses of these physiological systems, RVLM involvement in these age-related changes is not clear. Previous work using high-throughput gene expression analysis of the RVLM in aged animals suggested that chemical neurotransmission-related genes might be downregulated with advancing age. Since the RVLM function involves a balance of inhibitory and excitatory inputs, which is largely mediated by gamma-aminobutyric acid (GABA) and excitatory amino acid (EAA) neurotransmission, we hypothesized that aging is associated with altered excitatory and/or inhibitory neurotransmission-related gene expression in the RVLM. To test this hypothesis, we micropunched an RVLM-containing area from young (3–5 months), middle-aged (12–14 months), and aged (22–26 months) Fischer 344 male rats. RNA purified from these micropunches was analyzed using GABA and Glutamate RT2 Profiler PCR arrays (n= 8–10). In addition, the expression of selected genes was validated at the RNA level using TaqMan® based- qPCR and at the protein level using western blotting. All the genes that displayed significant differential expression (1.5-fold, p < .05, FDR < .05) were identified to be downregulated in the RVLM of aged and middle-aged rats compared to young rats. Among the downregulated genes, the percentage of glutamate neurotransmission-related genes was higher than GABA neurotransmission-related genes. Solute carrier family 1 member 6 (Slc1a6) gene showed the highest fold downregulation at the RNA level in the RVLM of aged compared to young rats, and its protein product, Excitatory amino acid transporter 4 (EAAT4), showed a downregulatory trend in the RVLM of aged and middle-aged rats. These results suggest that molecular constituents of both GABA and glutamate neurotransmission might be altered in the RVLM of aged and middle-aged rats, and the changes in glutamate neurotransmission might be more prominent. Investigating age-associated anatomical and functional changes in RVLM GABA and glutamate neurotransmission might provide a foundation for understanding the effects of aging on physiological function.
Project description:Transcriptomics reveal strain-specific metabolic strategies for acid resistance and gamma-aminobutyrate (GABA) production in Levilactobacillus brevis.
Project description:Succinate semialdehyde dehydrogenase (SSADH) deficiency is a rare autosomal recessive disorder effecting approximately 350 people around the world. Patients suffering from SSADH deficiency experience language acquisition failure, memory deficiencies, autism, increased aggressive behaviors, and seizures. There is a chemical buildup of both gamma-aminobutyric acid (GABA) and gamma-hydroxybutyric acid (GHB) in the neurological system of these patients. The Aldh5a1-/- knock out mouse model of SSADH deficiency shows the same chemical imbalances as the human disease, with additional fatal tonic-clonic seizures at three weeks of age. The elucidation of seizure causing pathways will facilitate treatment of seizure phenotypes in diseases with related epilepsy. ,Gene expression patterns within the hippocampus, cerebellum, and cortex of SSADH deficient mice (Aldh5a1-/- mice) will be compared to wild type mice at a time point immediately prior to fatal seizures. ,We hypothesis that the SSADH deficient mice experience a dysfunction of glutamate/GABA/ glutamine neurotransmitter cycle linked to astroglial metabolism and/or uptake of neuronally-released glutamate. The increased levels of GHB and GABA lead to down regulation of GABA-B-Receptor leading to seizures. The SSADH deficient phenotype may also be caused by ongoing oxidative damage and the pathological role of succinic semialdehyde.,SSADH deficient mice (Aldh5a1-/- knock out) exhibit fatal seizures around three weeks of age. Mutant and wild type mice will be sacrificed between 17 and 19 days of life, and brain sections will be extracted and frozen (using a standard protocol). Hippocampus, cerebellum, and cortex from three mutant mice and three wild type mice will individually be expression profiled on the Affymetrix platform, giving a total of eighteen arrays. Comparative analysis will then be carried out, evaluating the transcript differences between mutant and wild type mice in each brain region.
Project description:The presence of anti-microbial phenolic compounds, such as the model compound ferulic acid, in biomass hydrolysates poses significant challenges to the widespread use of biomass in conjunction with whole cell biocatalysis or fermentation. Biofuel toxicity must also be overcome to allow for efficient production of next generation biofuels such as butanol, isopropanol, and others for widespread usage. Currently, these inhibitory compounds must be removed through additional downstream processing or sufficiently diluted to create environments suitable for most industrially important microbial strains. This study explores the high ferulic acid and n-butanol tolerance in Lactobacillus brevis (L. brevis), a lactic acid bacteria often found in fermentation processes, by global transcriptional response analysis. The transcriptional profile of L. brevis under ferulic acid and butanol stress reveals that the presence of ferulic acid primarily triggers the expression of membrane proteins to counteract ferulic acid induced changes in membrane fluidity and ion leakage. In contrast to the ferulic acid stress response, butanol addition to growing cultures uniquely induced the entire fatty acid synthesis pathway in the midst of a generalized stress response. Overexpression of the rate-limiting acetyl-CoA carboxylase subunits (AccABCD) in E. coli to increase lipid synthesis had no effect on butanol tolerance, suggesting that additional engineering is necessary to produce sufficient levels of appropriate fatty acids to confer butanol tolerance. Several promising routes for understanding both phenolic acid and butanol tolerance have been identified based upon these findings. These insights may be used to guide further engineering of model industrial organisms to better tolerate both classes of inhibitors in processed biomass used for biofuel production.