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Background Bifidobacteria are gram-positive, probiotic, and generally regarded as safe bacteria. Techniques such as transformation, gene knockout, and heterologous gene expression have been established for  Bifidobacterium , indicating that this bacterium can be used as a cell factory platform. However, there are limited previous reports in this field, likely because of factors such as the highly anaerobic nature of this bacterium.  Bifidobacterium adolescentis  is among the most oxygen-sensitive  Bifidobacterium  species. It shows strain-specific gamma-aminobutyric acid (GABA) production. GABA is a potent bioactive compound with numerous physiological and psychological functions. In this study, we investigated whether  B. adolesentis  could be used for mass production of GABA. Results The  B. adolescentis  4–2 strain isolated from a healthy adult human produced approximately 14 mM GABA. It carried  gadB  and  gadC , which encode glutamate decarboxylase and glutamate GABA antiporter, respectively. We constructed pKKT427::P  ori   -gadBC  and pKKT427::P  gap   -gadBC  plasmids carrying  gadBC  driven by the original  gadB  ( ori ) and  gap  promoters, respectively. Recombinants of  Bifidobacterium  were then constructed. Two recombinants with high production abilities, monitored by two different promoters, were investigated. GABA production was improved by adjusting the fermentation parameters, including the substrate concentration, initial culture pH, and co-factor supplementation, using response surface methodology. The optimum initial cultivation pH varied when the promoter region was changed. The  ori  promoter was induced under acidic conditions (pH 5.2:4.4), whereas the constitutive  gap  promoter showed enhanced GABA production at pH 6.0. Fed-batch fermentation was used to validate the optimum fermentation parameters, in which approximately 415 mM GABA was produced. The conversion ratio of glutamate to GABA was 92–100%. Conclusion We report high GABA production in recombinant  B. adolescentis . This study provides a foundation for using  Bifidobacterium  as a cell factory platform for industrial production of GABA.

Cell factory for γ-aminobutyric acid (GABA) production using Bifidobacterium adolescentis