Latest revision as of 14:15, 12 October 2023

Bifunctional glutamate-cysteine ligase/glutathione synthase (gshF)

The BBa_K2856001 harbors a coding sequence of bi-functional glutamate--cysteine ligase/glutathione synthase (gshF) derived from S.agalactiae. Codon-optimization has been made for Lactococcus Lactis. gshFp catalyzes the conversion of Cys, Glu and Gly to GSH.

Usage and Biology

Bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) is an enzyme involved and responded to synthetic reaction of GSH. In this reaction, one Cysteine and one Glutamate are converted to one γ-GC, then one γ-GC and one Glycine are converted to one GSH (Figure 1). The Lactococcus Lactis NZ9000 has inability to synthesis GSH. In our project, we construct a plasmid harboring gshF in order to produce GSH in Lactococcus Lactis NZ9000.
[http://2018.igem.org/Team:H14Z1_Hangzhou Team H14Z1_Hangzhou 2018]

Figure. 1 Enzymatic reaction catalyzed by gshF

Construction and validation of plasmid pNZ-gshF

Gene gshF was amplified from genomic DNA of S. agalactiae and cut with restriction enzyme Hind III and NcoI, and ligased with plasmid pNZ8148 cut with the same enzyme. Then the ligation product was transferred to E.coli and spread on plates containing 10 mg/L chloramphenicol. Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. As shown in Figure. 2, all the picked colonies had gene gshF, illustrating that the plasmid pNZ-gshF was successfully constructed.

Figure. 2 Validation of plasmid pNZ-gshF. M represented marker. 1, 2 and 3 represented three randomly picked colonies.

Protein Analysis

After transferring the plasmid pNZ-gshF to L. lactis NZ9000, SDS-PAGE was performed to detect the protein expression level of gshF gene. The cells were washed twice with 0.1 M PBS after centrifugation. Crude protein was extracted through cell breaking using ultrasonication and centrifugation. Then the supernatant of the samples were used to analysis the protein expression. As shown in Figure. 3, expected bands of the GshF protein were observed on the gel in the lane of recombinant L. lactis containing pNZ-gshF induced with different nisin concentration while no GshF protein existed in L. lactis NZ9000.

Figure. 3 SDS-PAGE validation of gene gshF expression in L. lactis. M represented marker. WT represented L. lactis NZ9000. 1-3 represented L. lactis/pNZ-gshF induced with 100, 50 and 20 ng/ml nisin.

Validation of glutathione (GSH) by HPLC analysis

To confirm the synthetic glutathione in L. lactis/pNZ-gshF, HPLC was performed to analyze the extracts from the strain. Glutathione was identified on the basis of retention times related to standard sample. According to the retention time of standard glutathione sample, it can be confirmed that glutathione was synthesized in L. lactis/pNZ-gshF.

Figure. 4 Validation of glutathione (GSH) by HPLC.

BFSU-ICUnited 2023

Description

Hair damage can be triggered by environmental factors such as chemical treatment, heat, sunlight and pollution. Glutathione, as a natural antioxidant, can effectively neutralize free radicals and reduce free radical damage to hair. What's more, it can also act as a reducing agent, regulating disulfide bonds, thereby enhancing the elasticity and elasticity of hair.

Usage and Biology

To produce glutathione, we overexpress GshF in E. coli. Specifically, we used pLac promoter (Lactose promoter) to express gshF in pSB1A3 plasmid, and the recombinant plasmid was transformed into E. coli BL21.

Figure 1 Design of genetic circuit for gshF overexpression.

Figure 2 Gel electrophoresis of the gshF.

To measure the production of Glutathione (GSH), the engineered strain was resuspended in LB medium to an OD600 of 0.1 and incubated at 37°C for 2 hours. The GSH content was determined using a reduced form glutathione (GSH) detection kit, and the results are shown in Figure 3A. Initially, the strain was resuspended in LB medium to an OD600 of 0.1 and incubated at 37°C. Samples were taken at 4, 6, 8, and 12 hours to measure OD600 and assess the impact of GshF on bacterial growth. The results are shown in Figure 3B.

Additionally, different amounts of glutathione were added, and samples were taken at 4, 6, 8, and 12 hours to measure OD600 and assess the impact of glutathione on bacterial growth. The results are shown in Figure 3C. Furthermore, we investigated the effect of adding amino acid precursors (20 mM L-glutamic acid, L-cysteine, and glycine) on GSH production for 2 hours, and the results are shown in Figure 3D.

Characterization

Figure 3 Experimental results related to GshF.

To determine the enzyme activity of GshF, we cloned its coding gene sequence into pET28a vector and transformed it into E. coli BL21. The engineered bacteria were then cultured overnight in LB medium, and 0.5 mM IPTG was added for induction. The next day, 1 g of bacteria was collected by centrifugation and resuspended in PBS (pH 7.4). The cells were then sonicated (150 W, 1 s on, 3 s off, for a total of 20 minutes) to obtain cell lysate. The reaction mixture in 10 mL PBS contained 20 mM MgCl2, 20 mM ATP, 20 mM L-glutamine, 20 mM L-cysteine, and 20 mM glycine. After incubating at 37°C for 1 hour, the amount of reduced glutathione (GSH) was determined using a GSH assay kit, as shown in Figure 2E.

The effect of oxygen on GSH production was also tested (2 hours), as shown in Figure 2F, by culturing the engineered bacteria in a CO2 incubator with O2 concentration adjusted to 0%, 20%, and 30%. After incubating at 37°C for 2 hours, the amount of GSH was measured using a GSH assay kit. The results indicated that the engineered strain significantly enhanced GSH production, with the highest yield at an O2 concentration of 20%.

Potential application directions

The future construction of this engineered strain can help eliminate free radicals, reduce oxidative damage, and protect cells from oxidative stress, playing an antioxidant role. Glutathione has a regulatory effect on the immune system, enhancing the function of immune cells and promoting normal immune responses. It can also be used in industries such as food, pharmaceuticals, and cosmetics. Due to its multiple functions and potential for wide-ranging applications, glutathione has attracted significant attention in scientific research and product development.

References

Ask, Magnus, et al. "Engineering glutathione biosynthesis of Saccharomyces cerevisiae increases robustness to inhibitors in pretreated lignocellulosic materials." Microbial cell factories 12.1 (2013): 1-10.

Sequence and Features