Part:BBa_K3769002

gadB

Gene gadB produces an enzyme called GadB, which can catalyze the conversion of glutamate to GABA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 700
  
• 1000
  COMPATIBLE WITH RFC[1000]

Demonstrate the function of gadB

Gene gadB was amplified using E. coli genome as the template. We used the Golden Gate Assembly method to construct the following plasmid T7-T7RBS-gadB-T500, that is the gene gadB is under the control of the classic T7 promoter and its corresponding ribosome binding site, with a terminator T500. The plasmid was transformed into E. coli BL21(DE3) for expression. After induction with 100 μM IPTG, cells were lysed and GadB proteins were purified.

Fig 1. An SDS-PAGE gel of GadB protein purification. 50mmol/L, 100 mmol/L, 200 mmol/L, 500 mmol/L were samples collected when eluted using elution buffer with 50 mM imidazole, 100 mM imidazole, 200 mM imidazole, and 500 mM imidazole, respectively.

The enzymatic reaction was conducted in a 2 mL reaction mixture consisting of 200 mM Na2HPO4-citric acid buffer (pH 4.0), 50 mM L-MSG, 0.01 mM PLP, and 50–100 μL of purified enzyme. 1-fluoro-2,4-dinitrobenzene (FDNB) was then added to the reaction to get a yellow compound dinitroxine benzodiazepines (DNP amino acids) which has an absorbance at 485 nm. The mixtures were thoroughly mixed and incubated at 60°C for 1 hour. After cooling to room temperature, added PBS (0.02 mol/L, pH 7.0) to the mixtures to make a final volume of 10 mL. Then we measured the absorbance at 485 nm. The result was shown below, indicating that GABA was indeed generated in the reaction and the function of gadB was as expected.

Fig 2. Left: GABA production from GadB; Right: negative control.

Contribution From JIASHU-SouthChina

Description

γ-Aminobutyric acid (GABA) is a key inhibitory neurotransmitter in the central nervous system, known for its water solubility, thermal stability, and safety as an ingredient in food and beverages. Due to its anti-anxiety and stress-relieving properties, GABA is widely used in the food and health supplement industries. Given the need for an edible, anti-stress, and anti-anxiety compound in our project, we initiated research into the production of GABA.

Figure 1. GAD catalyzes glutamic acid to produce GABA.

Usage and Biology

We first designed and constructed a GABA production strain. The GadB gene (BBa_K3769002), encoding glutamate decarboxylase (GAD), was synthesized and codon-optimized for expression in E. coli. The gene was cloned into the pET23b plasmid using the EcoRI and XhoI restriction sites, generating the recombinant plasmid p23b-GadB. The construct was verified through sequencing, and the recombinant plasmid was extracted using a plasmid extraction kit. After verification, the plasmid was transformed into E. coli strains DH5α (for plasmid storage) and BL21 (for protein expression)(Figure 14).

Figure 2. Gene circuit of GadB.

Characterization

GABA verification

To catalyze GABA synthesis, we employed crude enzyme extracts from the engineered E. coli strains. The process involved the following steps: 1. Cell Harvesting and Enzyme Extraction: We collected 2 mL of bacterial culture, centrifuged it at 8000 rpm for 10 minutes to obtain the bacterial pellet. The pellet was resuspended in 2 mL of acetic acid-sodium acetate buffer (pH=4.6). This suspension was subjected to ultrasonic treatment (75 W, 1 second pulses with 3 second intervals, for a total of 20 minutes) in an ice bath to obtain the crude enzyme solution. 2. GABA Synthesis Reaction: In 1 mL of crude enzyme solution, 2% glutamic acid was added as the substrate, and the mixture was incubated at 37°C for 3 hours. 3. Quantification of GABA: After the reaction, GABA content was measured using a GABA test kit. The principle of the GABA test kit is that phenol and sodium hypochlorite react with GABA to produce a blue-green product, which has a maximum absorbance at 640 nm. Absorbance at 640 nm was recorded using a microplate reader, and a standard curve was generated to calculate the GABA concentration in the samples. Figure 4A indicates a strong linear relationship between GABA concentration and absorbance. Therefore, we conclude that GABA concentration can be reliably calculated based on absorbance. Figure 4B indicates that we can produce GABA through construct the p23b-GadB.

Figure 3. Agarose gel electrophoresis of GadB. The expected band is at 1398bp and the maker used was 2kb.

Figure 4. The production of GABA. (A) The standard curve of GABA. The linear regression equation is Y = 0.5130*X - 0.05257, with an R² value of 0.98. (B) The influence of inserting GadB gene fragments on GABA yield. BL21 and BL21/pET23b were used as control groups, while BL21/p23b-GadB served as the experimental group for the controlled experiments. By constructing the recombinant strain p23b-GadB, we successfully achieved GABA production, with the recombinant strain yielding 2.32 ± 0.21 g/L.

Effect of pH level on the activity of GadB

To enhance GadB enzyme activity, we tested the GABA concentration in the crude enzyme solution of the engineered strain under different pH conditions. The BL21/p23b-GadB pellet was resuspended in 2 mL of either acetate-sodium acetate buffer (pH=4.6), acetate-sodium acetate buffer (pH=3.6), PBS (pH=7.4), or Tris-HCl buffer (pH=9.2). The cells were then sonicated under ice bath conditions to obtain the crude enzyme solution. For each 1 mL of crude enzyme solution, 2% glutamic acid was added and incubated at 37°C for 3 hours. The GABA concentration was then measured using the γ-aminobutyric acid (GABA) detection kit (mlbio, China). The GadB enzyme shows optimal activity in acidic conditions, with the highest GABA production observed at pH= 4.6. Enzyme activity decreases significantly in neutral and alkaline environments, indicating that GadB functions best in an acidic environment.

Figure 5. The effect of pH on GadB activity.The activity of GadB enzyme varied significantly under different pH conditions. At pH=3.6 and pH=4.6, GABA production was higher, reaching 2.17 ± 0.12 g/L and 2.48 ± 0.23 g/L, respectively. In contrast, under neutral and alkaline conditions (pH=7.4 and pH=9.2), GABA production was significantly lower, with values of 0.61 ± 0.35 g/L at pH=7.4 and 0.03 ± 0.02 g/L at pH=9.2.

Potential application directions

This experiment demonstrates that the artificially synthesized GadB can be expressed normally in Escherichia coli, resulting in the successful production of GABA. Additionally, we optimized the production conditions, particularly the pH, with the highest GABA yield observed at pH=4.6. In the future, this technology can be applied to large-scale industrial production of GABA for use in functional foods, beverages, and health supplements. Furthermore, it could be expanded to pharmaceutical applications targeting anxiety and stress relief, offering a natural, safe alternative for mood regulation and mental health improvement.

Reference

Rashmi D, Zanan R, John S, et al. γ-aminobutyric acid (GABA): Biosynthesis, role, commercial production, and applications[J]. Studies in natural products chemistry, 2018, 57: 413-452.