Gamma-aminobutyric acid (GABA) plays a role in several physiological functions. GABA production by lactic acid bacteria has attracted considerable interest; however, there is need to improve production. This study aimed to develop a pH auto-sustain (PAS)-based GABA fermentation process for Lactobacillus brevis CD0817, with L-glutamic acid (solubility ~6.0 g/L and isoelectric point 3.22) as the substrate. Firstly, we determined the optimum levels of vital factors affecting GABA synthesis using Erlenmeyer flask experiments. The results showed that optimal levels of sugar, yeast extract, Tween-80, manganese ion, and temperature were 5.0 g/L, 35.0 g/L, 1.0 g/L, 16.0 mg/L, and 30.0 °C, respectively. The added L-glutamic acid (650 g per liter of medium) mostly existed in the form of solid powder was slowly released to supply the substrate and acidity essential for GABA production with the progress of fermentation. Based on the optimizations, the PAS-based GABA fermentation was performed using a 10 L fermenter. The PAS-based strategy promoted GABA synthesis by the strain of up to 321.9 ± 6.7 g/L after 48 h, with a productivity of 6.71 g/L/h and a substrate molar conversion rate of 99.6%. The findings suggest that the PAS-based fermentation is a promising method for GABA production by lactic acid bacteria.

Genome walking is a method used to retrieve unknown flanking DNA. Here, we reported wristwatch (WW) PCR, an efficient genome walking technique mediated by WW primers (WWPs). WWPs feature 5′- and 3′-overlap and a heterologous interval. Therefore, a wristwatch-like structure can be formed between WWPs under relatively low temperatures. Each WW-PCR set is composed of three nested (primary, secondary, and tertiary) PCRs individually performed by three WWPs. The WWP is arbitrarily annealed somewhere on the genome in the one low-stringency cycle of the primary PCR, or directionally to the previous WWP site in one reduced-stringency cycle of the secondary/tertiary PCR, producing a pool of single-stranded DNAs (ssDNAs). A target ssDNA incorporates a gene-specific primer (GSP) complementary at the 3′-end and the WWP at the 5′-end and thus can be exponentially amplified in the next high-stringency cycles. Nevertheless, a non-target ssDNA cannot be amplified as it lacks a perfect binding site for any primers. The practicability of the WW-PCR was validated by successfully accessing unknown regions flanking Lactobacillus brevis CD0817 glutamate decarboxylase gene and the hygromycin gene of rice. The WW-PCR is an attractive alternative to the existing genome walking techniques.

There is a recent trend of using lactic acid bacteria for the production of gamma-aminobutyric acid (GABA). This study described a method that combines fermentation and self-buffered whole-cell catalysis for the efficient production of GABA using Levilactobacillus brevis CD0817. Upon the completion of GABA fermentation, cells were recovered to conduct whole-cell catalysis by which the substrate L-glutamic acid was catalytically decarboxylated to GABA. L-glutamic acid itself maintained the acidity essential for decarboxylation. To maximize the whole-cell catalysis ability, the effects of the cell culture method, catalysis temperature, catalysis time, cell concentration, and L-glutamic acid dosage were investigated. The results illustrate that the cells that were cultivated for 16 h in a fermentation medium supplemented with 20.0 g/L of glucose were the most suitable for the whole-cell catalytic production of GABA. At 16 h, the fermentative GABA content reached 204.2 g/L. Under optimized whole-cell catalytic conditions (temperature 45.0 °C, time 12.0 h, wet cells 25.0 g/L, and L-glutamic acid 120.0 g/L), 85.1 g/L of GABA was obtained, with 3.7 ± 0.9 g/L of substrate residue. GABA was recovered from the system by sequentially performing rotary vacuum evaporation, precipitation with ethanol, filtration with filter paper, and drying. The purity of the GABA product reached 97.1%, with a recovery rate of 87.0%. These data suggest that the proposed method has potential applications in the production of GABA.