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

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::Pori-gadBC and pKKT427::Pgap-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.

Draft Genome Sequence of Bifidobacterium adolescentis 4-2, Isolated from Healthy Human Feces.

Bifidobacterium adolescentis 4-2 was isolated from healthy human feces. Here, we report a draft genome sequence of this bacterium, which may clarify the functionality of gut microbiota-brain communication. The draft genome comprises 2.39 Mb, with an average G+C content of 59.2% and 2,028 coding DNA sequences. An operon for gamma aminobutyric acid (GABA) biosynthesis was observed in the draft genome.

Bifidobacterium-Escherichia coli Shuttle Vector Series pKO403, with Temperature-Sensitive Replication Origin for Gene Knockout in Bifidobacterium.

A series of Bifidobacterium-Escherichia coli shuttle vectors (pKO403-lacZ'-Cm, pKO403-lacZ'-Sp, pKO403-lacZ'-p15A) were constructed based on the pKO403 backbone, which carries a temperature-sensitive replication origin. These vectors carry the lacZ'α fragment, overhung by two facing type IIS restriction sites, for blue-white selection and seamless gene cloning. These vectors are useful for gene knockout or multigene integration into the chromosome of Bifidobacterium.

Differences in the Concentration of the Fecal Neurotransmitters GABA and Glutamate Are Associated with Microbial Composition among Healthy Human Subjects.

Recent studies have shown that the gut microbiota modulates the physical and psychological functions of the host through several modes of action. One of them is mediating the production of active neurotransmitters, such as serotonin and gamma-aminobutyric acid (GABA). GABA is the major inhibitory neurotransmitter in the central nervous system. Here, we analyzed the relationship between fecal GABA concentration and microbial composition in more than 70 human participants. The gut microbiome composition was analyzed using next-generation sequencing based on 16S ribosomal RNA. High-performance liquid chromatography was used to evaluate the neurotransmitters GABA and glutamate. The GABA level was detected in a broad range (0-330 µg/g feces). The participants' samples were classified into high (>100 µg/g), medium (10-100 µg/g), and low (<10 µg/g) groups, based on fecal GABA concentration. The results reveal that the microbiome of the high-GABA samples had lower alpha diversity than the other samples. Beta diversity analysis showed significant (p < 0.05) separation between the high-GABA samples and others. Furthermore, we surveyed the abundance of specific GABA producer biomarkers among the microbiomes of tested samples. The family Bifidobacteriaceae exhibited high abundance in the microbiome of the high-GABA group. This study demonstrated that Bifidobacterium abundance was associated with high fecal GABA content in healthy human subjects. These results may aid the development of potential probiotics to improve microbial GABA production, which can support the maintenance of the physical and psychiatric health of the host.

A New Escherichia coli Entry Vector Series (pIIS18) for Seamless Gene Cloning Using Type IIS Restriction Enzymes.

A series of new Escherichia coli entry vectors (pIIS18-SapI, pIIS18-BsmBI, pIIS18-BsaI, pIIS18-BfuAI-1, and pIIS18-BfuAI-2) was constructed based on a modified pUC18 backbone, which carried newly designed multiple cloning sites, consisting of two facing type IIS enzyme cleavage sites and one blunt-end enzyme cleavage site. These vectors are useful for seamless gene cloning.