Team:BNDS China/Description
Project Description
The goal of our 2017 iGEM project is to genetically modify E. coli Nissle 1917, a probiotic, in such a way as to allow this organism to produce γ-Aminobutyric Acid (GABA). The three key words of our project are: GABA, GAD, and E. coli Nissle 1917.
I. GABA
GABA is a four carbon non-protein amino acid that functions as an inhibitory neurotransmitter in the brain, affecting one’s personality and stress management ability. AM et. al tested sixty-three adults with capsules containing 100 mg of GABA or dextrin as a placebo, and found that after 30 min, the experimental group had a decrease in the alpha and beta band waves compared to the control group, indicating that GABA might have alleviated the stress induced by mental tasks during the experiment. Moreover, GABA may be able to treat diabetes, according to Watanabe M, 2017. Clinically, GABA is used for treating sleeplessness, depression, autonomic disorders, chronic alcohol-related symptoms, and stimulation of immune cells ,(Siragusa S et al., 2007). Besides these uses, GABA is also sold on the market as an over the counter dietary supplement.
Fig. 1. Two mechanisms of GABA in CNS
Though GABA is beneficial to human beings in many ways, it is not allowed to be produced through chemical process if the chemical is sold as an edible product since there may be harmful impurities during the production. Therefore, it becomes meaningful for us to use synthetic biology methods to produce food grade GABA.
II. Glutamate Decarboxylase (GAD)
Many microorganisms, including some Escherichia coli and Lactobacillus strains, can produce GABA (Dhakal et al.). Among these microorganisms, Lactobacillus brevis NCL912 has shown the highest production of GABA: 3.57 g/l. According to Dhakal et al., the difference in productivity of GABA in different strains is caused by the difference in their GAD enzymes’ properties, suggesting that the strain Lactobacillus brevis NCL912 may possess a unique GAD system, incorporating a unique GAD gene.
Li et al. characterized the components of GAD operon in Lactobacillus NCL912 in 2013: gadC, gadA, and gadR. GadA is the glutamate decarboxylase that catalyzes the chemical reaction from glutamate to GABA, while gadC functions as a glutamate/GABA antiporter, an exchanger of GABA and glutamate. Typically, gadC is only present in the genomes of limited strains indicating their strain-specific characteristic of GABA production (Wu et al., 2017). GadCA forms an operon and is regulated by gadR, which had much higher expression levels (Li, Li, Liu & Cao, 2013).
For the outstanding GABA producing ability of the GAD system in Lactobacillus brevis NCL912, we picked proteins from this system as components of our device.
III. E. coli Nissle 1917
E. coli Nissle 1917, a harmless strain of E. coli isolated by Alfred Nissle, is a commonly used probiotic in treating Ulcerative colitis, chronic constipation, and Crohn's disease. We chose this strain as our genetic engineering project due to its safety and its controllability. Since many researches have shown that E. coli Nissle 1917 is edible, we are able to derive our product, GABA containing milk, directly from the genetically modified species. Moreover, E. coli Nissle can be transformed and cultured using the same techniques as that of other E. coli strains.
Thus, by inserting the sequence of unique GAD system found in Lactobacillus brevis NCL912 into E. coli Nissle, we designed multiple parts and tested their individual abilities.
References
Abdou AM, Higashiguchi S, Horie K, Kim M, Hatta H, Yokogoshi H. (2006).Relaxation and immunity enhancement effects of gamma-aminobutyric acid (GABA) administration in humans [abstract]. BoiFactor. 26(3):201-8. doi: 10.1002/biof.5520260305
Dhakal, Radhika., Bajpai, Vivek K., Baek, Kwang-Hyun. (2012, Jun 7). Production of gaba (γ - Aminobutyric acid) by microorganisms: a review. Brazilian Journal of Microbiology, 1230-1241. doi: http://dx.doi.org/10.1590/S1517-83822012000400001
Li H, Cao Y, Gao D, Xu H. (2008, Dec.). A high γ-aminobutyric acid-producing ability Lactobacillus brevis isolated from Chinese traditional paocai. Ann Microbiol, 58, 649–653. doi: 10.1007/BF03175570
Li H, Li W, Liu X & Cao Y. (2013 Nov. 6). gadA gene locus in Lactobacillus brevis NCL912 and its expression during fed-batch fermentation. FEMS Microbiol Lett, 349 (2013) 108–116 doi: 10.1111/1574-6968.12301
Siragusa S., De Angelis M., Di Cagno R., Rizzello CG., Coda R., Gobbetti M. (2007 Nov.). Synthesis of γ-aminobutyric acid by lactic acid bacteria isolated from a variety of Italian cheeses. Appl Environ Microbiol, 73(22), 7283–7290. doi: 10.1128/AEM.01064-07.
Wu Q, Tun HM, Law YS, Khafipour E, Shah NP. (2017 Feb.). Common Distribution of gad Operon in Lactobacillus brevis and its GadA Contributes to Efficient GABA Synthesis toward Cytosolic Near-Neutral pH. Front Microbiol. 2017; 8: 206. doi: 10.3389/fmicb.2017.00206
