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.

Structure of a Core Promoter in Bifidobacterium longum NCC2705.

Bacterial promoters consist of core sequence motifs termed -35 and -10 boxes. The consensus motifs are TTGACA and TATAAT, respectively, which were identified from leading investigations on Escherichia coli However, the consensus sequences are not likely to fit genetically divergent bacteria. The sigma factor of the genus Bifidobacterium has a characteristic polar domain in the N terminus, suggesting the possibility of specific promoter recognition. We reevaluated the structure of Bifidobacterium longum NCC2705 promoters and compared them to other bacteria. Transcriptional start sites (TSSs) of the B. longum NCC2705 strain were identified using transcriptome sequencing (RNA-Seq) analysis to extract promoter regions. Conserved motifs of a bifidobacterial promoter were determined using regions upstream of TSSs and a hidden Markov model. As a result, consensus motifs of the -35 and -10 boxes were TTGTGC and TACAAT, respectively. To assess each base of both motifs, we constructed 37 plasmids based on pKO403-TPCTcon, including the hup promoter connected with a chloramphenicol acetyltransferase as a reporter gene. This reporter assay showed two optimal motifs of the -35 and -10 boxes, namely, TTGNNN and TANNNT, respectively. We further analyzed spacer lengths between the -35 and -10 boxes via a bioinformatics approach. The spacer lengths predominant in bacteria have been generally reported to be approximately 17 bp. In contrast, the predominant spacer lengths in the genus Bifidobacterium and related species were 11 bp, in addition to 17 bp. A reporter assay to assess the spacer lengths indicated that the 11-bp spacer length produced unusually high activity.IMPORTANCE The structures of sigma factors vary among bacterial strains, indicating that recognition rules may also vary. Therefore, we investigated the promoter structure of Bifidobacterium longum NCC2705 using a bioinformatics approach and wet analyses. The most frequent and optimal motifs were similar to other bacterial consensus motifs. The optimal spacer length between the two boxes was reported to be 17 bp. It is widely applied to a bioinformatics approach for other bacteria. Unexpectedly, conserved spacer lengths were 11 bp as well as 17 bp in the genus Bifidobacterium Moreover, the sigma factor of the genus Bifidobacterium has a characteristic domain in the N terminus which may contribute to the additional functions. Hence, it would be valuable to reevaluate the promoter in other organisms.

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.

Construction of a reporter system for bifidobacteria using chloramphenicol acetyltransferase and its application for evaluation of promoters and terminators.

A reporter assay system is an essential tool for investigating gene expression mechanisms. In the case of bifidobacteria, several convenient and sensitive reporter systems have been developed. Here, we developed a new reporter system for bifidobacteria using the chloramphenicol acetyltransferase gene (cat) from Staphylococcus aureus. This enzyme stoichiometrically produced free CoA-SH, which was analyzed quantitatively with Ellman's test using 2-nitrobenzoic acid (DTNB). The 2-nitro-5-thiobenzoate (TNB2-) produced showed a strong yellowish color with maximum absorbance at 412 nm. We also constructed a new pBCMAT plasmid series for CAT assays in bifidobacteria to evaluate promoters and terminators. Analyses using promoters from Bifidobacterium longum NCC2705 indicated that the CAT assay using these promoters is quantitative, has a wide measurement range, and is stable. In addition, this assay was useful for several bifidobacterial species, including B. longum, Bifidobacterium breve, and Bifidobacterium adolescentis. Compared with evoglow-Bs2, a fluorescent protein used under anaerobic conditions, the CAT assay showed about 0.25% background activity. In analyses using this CAT assay, we identified 11 promoters and 12 terminators of B. longum NCC2705. The genes encoding ribosomal proteins, elongation factors, and transfer RNAs possessed strong promoters, and terminators that include strong stem-loops and poly-U tails structures tended to show high activities. Although the abovementioned promoters made stronger contributions to expression activities than the terminators, the maximum fold difference in the activities among the tested terminators was approximately 17-fold. Modification of the -10 box and 5'-UTR in the promoters and the structure around the stem-loop in the terminators affected expression levels. These results suggest that the CAT assay is useful for various analyses of bifidobacterial gene expression.

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.