Cell factory for γ-aminobutyric acid (GABA) production using <i>Bifidobacterium adolescentis</i>

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Abstract

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.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12934-021-01729-6.

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Most cited references 60

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A One Pot, One Step, Precision Cloning Method with High Throughput Capability

Carola Engler, Romy Kandzia, Sylvestre Marillonnet (2008)

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation.

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Is Open Access

Statistical analysis of real-time PCR data

Joshua S Yuan, Ann Reed, Feng Chen … (2006)

Background Even though real-time PCR has been broadly applied in biomedical sciences, data processing procedures for the analysis of quantitative real-time PCR are still lacking; specifically in the realm of appropriate statistical treatment. Confidence interval and statistical significance considerations are not explicit in many of the current data analysis approaches. Based on the standard curve method and other useful data analysis methods, we present and compare four statistical approaches and models for the analysis of real-time PCR data. Results In the first approach, a multiple regression analysis model was developed to derive ΔΔCt from estimation of interaction of gene and treatment effects. In the second approach, an ANCOVA (analysis of covariance) model was proposed, and the ΔΔCt can be derived from analysis of effects of variables. The other two models involve calculation ΔCt followed by a two group t-test and non-parametric analogous Wilcoxon test. SAS programs were developed for all four models and data output for analysis of a sample set are presented. In addition, a data quality control model was developed and implemented using SAS. Conclusion Practical statistical solutions with SAS programs were developed for real-time PCR data and a sample dataset was analyzed with the SAS programs. The analysis using the various models and programs yielded similar results. Data quality control and analysis procedures presented here provide statistical elements for the estimation of the relative expression of genes using real-time PCR.

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Metabolism and functions of gamma-aminobutyric acid.

B. Shelp (1999)

Gamma-aminobutyric acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. In plants, stress initiates a signal-transduction pathway, in which increased cytosolic Ca2+ activates Ca2+/calmodulin-dependent glutamate decarboxylase activity and GABA synthesis. Elevated H+ and substrate levels can also stimulate glutamate decarboxylase activity. GABA accumulation probably is mediated primarily by glutamate decarboxylase. However, more information is needed concerning the control of the catabolic mitochondrial enzymes (GABA transaminase and succinic semialdehyde dehydrogenase) and the intracellular and intercellular transport of GABA. Experimental evidence supports the involvement of GABA synthesis in pH regulation, nitrogen storage, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization. There is a need to identify the genes of enzymes involved in GABA metabolism, and to generate mutants with which to elucidate the physiological function(s) of GABA in plants.

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Author and article information

Contributors

Hend Altaib:

ORCID: http://orcid.org/0000-0003-1359-3637

hend_altaib@yahoo.com

Journal

Journal ID (nlm-ta): Microb Cell Fact

Journal ID (iso-abbrev): Microb Cell Fact

Title: Microbial Cell Factories

Publisher: BioMed Central (London )

ISSN (Electronic): 1475-2859

Publication date (Electronic): 7 March 2022

Publication date PMC-release: 7 March 2022

Publication date Collection: 2022

Volume: 21

Electronic Location Identifier: 33

Affiliations

[1 ]GRID grid.256342.4, ISNI 0000 0004 0370 4927, Laboratory of Genome Microbiology, Faculty of Applied Biological Sciences, , Gifu University, ; 1-1 Yanagido, Gifu, 501-1193 Japan

[2 ]GRID grid.256342.4, ISNI 0000 0004 0370 4927, The United Graduate School of Agricultural Science, , Gifu University, ; 1-1 Yanagido, Gifu, 501- 1193 Japan

[3 ]GRID grid.449014.c, ISNI 0000 0004 0583 5330, Department of Animal Medicine, Faculty of Veterinary Medicine, , Damanhour University, ; El-Beheira, Egypt

[4 ]GRID grid.7155.6, ISNI 0000 0001 2260 6941, Department of Pesticide Chemistry and Technology, Faculty of Agriculture, , Alexandria University, ; El-Shatby, Alexandria, 21545 Egypt

[5 ]GRID grid.256342.4, ISNI 0000 0004 0370 4927, Graduate School of Engineering, , Gifu University, ; 1-1 Yanagido, Gifu, 501-1193 Japan

Author information

Hend Altaib http://orcid.org/0000-0003-1359-3637

Article

Publisher ID: 1729

DOI: 10.1186/s12934-021-01729-6

PMC ID: 8903651

PubMed ID: 35255900

SO-VID: 9ca806b8-922b-4b0e-b1f8-a1865f03ad86

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

History

Date received : 2 September 2021

Date accepted : 20 December 2021

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100001691, Japan Society for the Promotion of Science;

Award ID: 16H04896

Award Recipient : Tohru Suzuki

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ScienceOpen disciplines: Biotechnology

Keywords: bifidobacterium adolescetis,gaba,fed batch fermentation

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ScienceOpen disciplines: Biotechnology

Keywords: bifidobacterium adolescetis, gaba, fed batch fermentation

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

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Abstract

Background

Results

Conclusion

Supplementary Information

Related collections

Genome Engineering using CRISPR

Most cited references 60

A One Pot, One Step, Precision Cloning Method with High Throughput Capability

Statistical analysis of real-time PCR data

Metabolism and functions of gamma-aminobutyric acid.

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Cited by 8

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