Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH

Abstract

Many strains of lactic acid bacteria (LAB) and bifidobacteria have exhibited strain-specific capacity to produce γ-aminobutyric acid (GABA) via their glutamic acid decarboxylase (GAD) system, which is one of amino acid-dependent acid resistance (AR) systems in bacteria. However, the linkage between bacterial AR and GABA production capacity has not been well established. Meanwhile, limited evidence has been provided to the global diversity of GABA-producing LAB and bifidobacteria, and their mechanisms of efficient GABA synthesis. In this study, genomic survey identified common distribution of gad operon-encoded GAD system in Lactobacillus brevis for its GABA production among varying species of LAB and bifidobacteria. Importantly, among four commonly distributed amino acid-dependent AR systems in Lb. brevis, its GAD system was a major contributor to maintain cytosolic pH homeostasis by consuming protons via GABA synthesis. This highlights that Lb. brevis applies GAD system as the main strategy against extracellular and intracellular acidification demonstrating its high capacity of GABA production. In addition, the abundant GadA retained its activity toward near-neutral pH (pH 5.5–6.5) of cytosolic acidity thus contributing to efficient GABA synthesis in Lb. brevis. This is the first global report illustrating species-specific characteristic and mechanism of efficient GABA synthesis in Lb. brevis.

Production of Gamma-Aminobutyric Acid from Lactic Acid Bacteria: A Systematic Review

“…The biosynthesis of GABA by microorganisms is performed by the glutamic acid decarboxylase (GAD, EC 4.1.1.15) system, which is composed of the GAD enzyme (encoded by gadA or gadB) and glutamate/GABA antiporter GadC [29][30][31][32][33]. The biosynthetic pathway of GABA by microbes is shown in Figure 1.…”

Screening Potential Probiotic Characteristics of Lactobacillus brevis Strains In Vitro and Intervention Effect on Type I Diabetes In Vivo

“…These results were closely related to the findings by Hiraga et al [ 32 ] who suggested that the gad gene of Lactobacillus brevis IFO 12005 was 1,440 bp. Moreover, Wu et al [ 19 ] reported that biochemical analysis and genetic screening have confirmed the common existence of gad system in Lactobacillus brevis suggesting its species-specific characteristic of GABA production.…”

“…The obtained results were consistent with those of Zhang [ 46 ], who showed the performance of antiacids of isolated strains. In addition, Wu et al [ 19 ] reported that pancreatic fluid had no significant effect on survival of LAB.…”

“…Genomic DNA was used as a template for PCR amplification. Fifty microliters of each PCR mixture contained DNA template 1.0 μ L, two primer pairs (ComateBio Custom Primers, Jilin, Changchun, China), gad -F/ gad R (10 μ mol / L) 2.0 μ L, [ 19 ] DNA Polymerase (2.5 U / μ L) 0.5 μ L, 10× Taq Buffer 5.0 μ L, 2'-deoxynucleoside 5' triphosphate (dNTPs) (2.5 mM) 4.0 μ L, and ddH 2 O 35.5 μ L. Furthermore, PCR amplification was performed using the GeneAmp PCR System 9700 thermal cycler (Applied Biosystem, USA) with the thermal cycling parameters as follows: denaturation at 95°C for 5 min, 30 cycles of 95°C for 30 s, 55°C for 1.30 min and 72°C for 1.30 min, with a final extension at 72°C for 10 min. A 5- μ L volume of PCR product was electrophoresed on a 1% agarose gel (Gibco BRL, France) at 90 V for 1.5 hours and then stained with ethidium bromide and band patterns were visualized.…”

Ribosomal binding site sequences and promoters for expressing glutamate decarboxylase and producing γ-aminobutyrate in Corynebacterium glutamicum

“…GABA is mainly converted from l -glutamate (Glu) by lactic acid bacteria (LAB) and Escherichia coli strains with high Glu decarboxylase (GAD) activity (Li and Cao 2010 ; Shi et al 2016 ). Although production of GABA by LAB, such as Lactobacillus brevis (Shi et al 2017b ; Wu et al 2017 ), is recognized as safe, the cost of production is high; whereas production of GABA by E. coli is high yield (Huang et al 2016 ; Zhao et al 2016a ) but is not acceptable for food usage. Furthermore, Glu must be added as the precursor during GABA production by LAB and E. coli , making such bioconversion not cost-effective.…”