Background In lactic acid bacteria (LAB), acid stress leads to decreases of cell vitality and fermentation yield. Glutamate decarboxylase (GAD) system is regarded as one of the essential acid-resistance mechanisms in LAB. However, the regulation of GAD system is not well identified in the genus Lactobacillus . Although potential transcriptional regulator gene located upstream of GAD system genes was found in several Lactobacillus species, such as Lactobacillus ( L. ) brevis , the contribution of the regulator to acid resistance of the genus Lactobacillus has not been experimentally determined. Results The potential transcriptional regulator gene gad R was disrupted by homologous recombination in L. brevis ATCC 367, leading to the decreased expression of gad C and gadB . The inactivation of GadR completely eliminated γ-aminobutyric acid (GABA) production and decreased the glutamate-dependent acid resistance. Moreover, expression of gad C and gad B in the presence of glutamate was increased and glutamate also stimulated the expression of gad R. In addition, L. brevis D17, a strain screened from acidic fermented grains of Chinese liquor production, had much higher expression level of gad R than the typical strain L. brevis ATCC 367. Under the pH-controlled and mixed-feed fermentation, L. brevis D17 achieved a titer of 177.74 g/L and a productivity of 4.94 g/L/h of GABA within 36 h. However, the L. brevis ATCC 367 only achieved a titer of 6.44 g/L and 0.18 g/L/h of GABA although the same fermentation control approach was employed. Conclusions GadR is a positive transcriptional regulator controlling GABA conversion and acid resistance in L. brevis . L. brevis strains with hyper-expressing of gad R are excellent candidates for GABA production in industrial scale. Electronic supplementary material The online version of this article (10.1186/s12934-019-1157-2) contains supplementary material, which is available to authorized users.

Lactic acid bacteria often encounter a variety of multiple stresses in their natural and industrial fermentation environments. The glutamate decarboxylase (GAD) system is one of the most important acid resistance systems in lactic acid bacteria. In this study, we demonstrated that GlnR, a nitrogen regulator in Gram-positive bacteria, directly modulated γ-aminobutyric acid (GABA) conversion from glutamate and was involved in glutamate-dependent acid resistance in Lactobacillus brevis. The glnR deletion strain (ΔglnR mutant) achieved a titer of 284.7 g/liter GABA, which is 9.8-fold higher than that of the wild-type strain. The cell survival of the glnR deletion strain was significantly higher than that of the wild-type strain under the condition of acid challenge and was positively correlated with initial glutamate concentration and GABA production. Quantitative reverse transcription-PCR assays demonstrated that GlnR inhibited the transcription of the glutamate decarboxylase-encoding gene (gadB), glutamate/GABA antiporter-encoding gene (gadC), glutamine synthetase-encoding gene (glnA), and specific transcriptional regulator-encoding gene (gadR) involved in gadCB operon regulation. Moreover, GABA production and glutamate-dependent acid resistance were absolutely abolished in the gadR glnR deletion strain. Electrophoretic mobility shift and DNase I footprinting assays revealed that GlnR directly bound to the 5′-untranslated regions of the gadR gene and gadCB operon, thus inhibiting their transcription. These results revealed a novel regulatory mechanism of GlnR on glutamate-dependent acid resistance in Lactobacillus. IMPORTANCE Free-living lactic acid bacteria often encounter acid stresses because of their organic acid-producing features. Several acid resistance mechanisms, such as the glutamate decarboxylase system, F1Fo-ATPase proton pump, and alkali production, are usually employed to relieve growth inhibition caused by acids. The glutamate decarboxylase system is vital for GAD-containing lactic acid bacteria to protect cells from DNA damage, enzyme inactivation, and product yield loss in acidic habitats. In this study, we found that a MerR-type regulator, GlnR, was involved in glutamate-dependent acid resistance by directly regulating the transcription of the gadR gene and gadCB operon, resulting in an inhibition of GABA conversion from glutamate in L. brevis. This study represents a novel mechanism for GlnR's regulation of glutamate-dependent acid resistance and also provides a simple and novel strategy to engineer Lactobacillus strains to elevate their acid resistance as well as GABA conversion from glutamate.

γ-Aminobutyric acid (GABA) is a ubiquitous nonprotein amino acid that has multiple physiological functions and has received significant attention in the pharmaceutical and food industries. Although there are many GABA-producing bacteria, the high cost of strain cultivation limits its food additive and pharmaceutical raw material application. In our study, Lactobacillus hilgardii GZ2, a novel GABA-producing strain, was investigated. We attempted to replace nitrogen sources with silkworm pupae, the waste resource of the silk reeling industry, in GYP complex medium. The GABA titer reached 33.2 g/L by using 10 g/L silkworm pupae meal instead of tryptone. Meanwhile, the pH of fermentation was automatically controlled by adjusting the addition of glucose and monosodium glutamate. Finally, the highest GABA yield and productivity were 229.3 g/L and 3.2 g/L/h in L. hilgardii when silkworm pupae meal was replaced with tryptone combined with glucose and monosodium glutamate feeding. By utilizing the waste resource to reduce the cost of the nitrogen source and automatically controlling the pH in L. hilgardii, a hyper titer and productivity of GABA was generated for applications in the food and pharmaceutical industries.