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United States Department of Agriculture

Agricultural Research Service

Research Project: FUNCTIONAL FOOD DEVELOPMENT BY MICROBIAL BIOTECHNOLOGY

Location: Dairy and Functional Foods

Title: Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus

Authors
item Renye, John
item Somkuti, George

Submitted to: Biotechnology Letters
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 19, 2011
Publication Date: March 1, 2012
Citation: Renye Jr, J.A., Somkuti, G.A. 2012. Vector-mediated chromosomal integration of the glutamate decarboxylase gene in streptococcus thermophilus. Biotechnology Letters. 34:549-555.

Interpretive Summary: Gamma-aminobutyric acid (GABA) is a potent neurotransmitter shown to induce relaxation, reduce anxiety, and lower blood pressure in humans. Some lactic acid bacteria (LAB), commonly used in the production of dairy foods, have been shown to naturally produce GABA and are considered potential probiotics for use in the development of functional foods. However, GABA production is limited to specific strains of these bacteria, thus the use of microbial biotechnology was investigated for its potential to transfer of this activity to non-producing LAB. In this study, the gene which encodes glutamate decarboxylase, an enzyme essential for the production of GABA, was transferred to a strain of Streptococcus thermophilus that does not produce GABA. Following transfer of the gene, it was shown that the newly developed bacterium produced amounts of GABA similar to those observed from a known GABA producing strain of S. thermophilus. This method of transfer is expected to allow the bacterium to maintain its food-grade status, while improving its potential to serve as a probiotic for improving the health-promoting properties of dairy foods.

Technical Abstract: The integrative vector pINTRS was used to transfer glutamate decarboxylase (GAD) activity to Streptococcus thermophilus ST128, thus allowing for the production of '-aminobutyric acid (GABA). In pINTRS, the gene encoding glutamate decarboxylase, gadB, was flanked by DNA fragments homologous to a S. thermophilus pseudogene to allow for integration at a non-essential locus on the chromosome. Screening techniques confirmed the insertion of gadB with either its endogenous promoter or the S. thermophilus P2201 promoter, resulting in the generation of recombinant strains, ST128/gadB or ST128/P2201-gadB. Following the integration event it was shown that unwanted plasmid DNA, specifically the erythromycin resistance gene, was eliminated from the recombinant strains. Based on the production of GABA, both strains displayed similar levels of GAD activity ( 23.0 – 23.8 uM/mg of dry cell weight).

Last Modified: 8/30/2014
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