Samira Sadat Taheri - Islamic Azad University of Damghan, Damghan, Iran
Mohammad Shafiee - Departmant of Medical Genetics, Golestan University of Medical Sciences, Gorgan, Iran.Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran
Ayyoob Khosravi - Department of Molecular Medicine, Faculty of Advanced Medical Technologies, Golestan University of Medical Sciences, Gorgan, Iran. Stem Cell Research Center, Golestan University of Medical Sciences, Gorgan, Iran
Marie Saghaeian - Biochemistry and Metabolic Disorders Research Center, Golestan University of Medical Sciences, Gorgan, Iran

Background: Gamma-Amino butyric acid (GABA) is an inhibitory neurotransmitter produced by irreversible decarboxylation of the glutamate by the glutamic acid decarboxylase (GAD) enzyme. It has been shown that GABA can be useful in colorectal cancer prevention. Here, we aimed to construct a recombinant Bifidobacterium animalis-lactis (BB-12) to produce GABA as a potential probiotic supplement.Materials and methods: The glutamate decarboxylase gene was synthesized and propagated in the pET-32a (+) expression vector. The plasmid was then purified and sub-cloned to BB-12 by electroporation. The E.coli was also transformed with recombinant plasmid by cold CaCl2 method as positive control. The recombinant colonies were confirmed by Colony-PCR and finally the culture medium was used for final determination of the GABA by HPLC.Results: Our findings showed that electroporation delivery of the recombinant GAD-pET-32a (+) to the BB-12 can result to GABA production in the culture medium after 72h of incubation equal to 19.774 ppm.Conclusion: Coloning of GAD gene was performed for the first time in this bacterium.The recombinant BB-12 encoding GAD can produce GABA which maybe useful as a genetically modified probiotic bacteria for local delivery of GABA in gut microbiome. However, further studies are needed.

Gamma-Amino butyric acid (GABA), Glutamate decarboxylase, Bifidobacterium animalis – Lactis, Probiotic