Abstract
BackgroundThe glutamate decarboxylase (GAD) system of Lactobacillus brevis involves two isoforms of GAD, GadA and GadB, which catalyze the conversion of L-glutamate to γ-aminobutyric acid (GABA) in a proton-consuming reaction contributing to intracellular pH homeostasis. However, direct experimental evidence for detailed contributions of gad genes to acid tolerance and GABA production is lacking.ResultsMolecular analysis revealed that gadB is cotranscribed in tandem with upstream gadC, and that expression of gadCB is greatly upregulated in response to low ambient pH when cells enter the late exponential growth phase. In contrast, gadA is located away from the other gad genes, and its expression was consistently lower and not induced by mild acid treatment. Analysis of deletion mutations in the gad genes of L. brevis demonstrated a decrease in the level of GAD activity and a concomitant decrease in acid resistance in the order of wild-type> ΔgadA> ΔgadB> ΔgadC> ΔgadAB, indicating that the GAD activity mainly endowed by GadB rather than GadA is an indispensable step in the GadCB mediated acid resistance of this organism. Moreover, engineered strains with higher GAD activities were constructed by overexpressing key GAD system genes. With the proposed two-stage pH and temperature control fed-batch fermentation strategy, GABA production by the engineered strain L. brevis 9530: pNZ8148-gadBC continuously increased reaching a high level of 104.38 ± 3.47 g/L at 72 h.ConclusionsThis is the first report of the detailed contribution of gad genes to acid tolerance and GABA production in L. brevis. Enhanced production of GABA by engineered L. brevis was achieved, and the resulting GABA level was one of the highest among lactic acid bacterial species grown in batch or fed-batch culture.
Highlights
The glutamate decarboxylase (GAD) system of Lactobacillus brevis involves two isoforms of GAD, GadA and GadB, which catalyze the conversion of L-glutamate to γ-aminobutyric acid (GABA) in a proton-consuming reaction contributing to intracellular pH homeostasis
To address the above issues, in the present work we focused our attention on the high GABA-producing strain L. brevis CGMCC1306 [21, 28, 29], with the aim of identifying genes that may explain its ability to grow under acid conditions and its GABA production
This is a common characteristic shared by L. brevis strains (Fig. 2), except for L. brevis NCL912, with only one cloned gene encoding GAD [27]
Summary
The glutamate decarboxylase (GAD) system of Lactobacillus brevis involves two isoforms of GAD, GadA and GadB, which catalyze the conversion of L-glutamate to γ-aminobutyric acid (GABA) in a proton-consuming reaction contributing to intracellular pH homeostasis. The growth of LAB is characterized by the generation of acidic end products of fermentation, which accumulate in the extracellular environment, lowering the pH and preventing the growth of spoilage bacteria [2, 3] This distinctive feature is the basis of widely practiced methods of food preservation via fermentation [4]. Among various types of acid responses and tolerance mechanisms, the GAD system is regarded as one of the most potent acid mitigating pathways In this system, intracellular protons are consumed through decarboxylation of glutamate in the cytoplasm and exchange of the reaction product GABA with extracellular glutamate, which contributes to protecting cells from the acid stress encountered during food fermentation and in the gastrointestinal tract [9, 10]
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