gshF

Description

gshF encodes a bifunctional glutathione synthetase GshF originated from Streptococcus thermophilus.

Usage

It is less sensitive to GSH, is and codon optimized for expressing in G. hansenii.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 178
Illegal EcoRI site found at 760
Illegal EcoRI site found at 1195
Illegal EcoRI site found at 1693
Illegal PstI site found at 19
Illegal PstI site found at 49
Illegal PstI site found at 406
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 178
Illegal EcoRI site found at 760
Illegal EcoRI site found at 1195
Illegal EcoRI site found at 1693
Illegal PstI site found at 19
Illegal PstI site found at 49
Illegal PstI site found at 406
Illegal NotI site found at 1838
Illegal NotI site found at 2038
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 178
Illegal EcoRI site found at 760
Illegal EcoRI site found at 1195
Illegal EcoRI site found at 1693
Illegal XhoI site found at 2019
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 178
Illegal EcoRI site found at 760
Illegal EcoRI site found at 1195
Illegal EcoRI site found at 1693
Illegal PstI site found at 19
Illegal PstI site found at 49
Illegal PstI site found at 406
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 178
Illegal EcoRI site found at 760
Illegal EcoRI site found at 1195
Illegal EcoRI site found at 1693
Illegal PstI site found at 19
Illegal PstI site found at 49
Illegal PstI site found at 406
Illegal NgoMIV site found at 1620
Illegal NgoMIV site found at 2251
1000
COMPATIBLE WITH RFC[1000]

2022 SZPT-China

1.Characterization in E. coli TOP10

As shown in Figure 2, the expression of GshF which encoded by BBa_K4325003 were verified successfully by PCR amplification and western blot respectively. The GSH production of E. coli TOP10 containing this part is much higher (~92 fold) than that of wild-type E. coli TOP10.

Figure 2: (a) Verification of gshF in E. coli TOP10; (b) Verification of western blot electrophoresis in E. coli Top10; (c) Comparison of GSH production between wild type and engineered of E. coli Top10.

2.Characterization in G. hansenii ATCC53582

Figure3 (a) shows the DNA fragments of gshF were amplified from G. hansenii, thus confirming the successful incorporation of the plasmid. Figure2 (b) shows that G. hansenii containing this part exhibited enhanced GSH biosynthesis, as evidenced by colorimetric analysis of glutathione.

Figure 3: (a) Verification of gshF in G. hansenii ATCC53582; (b) Comparison of GSH production between wild type and engineered G. hansenii.

References

[1] Li, W., Li, Z., Yang, J. & Ye, Q. Production of glutathione using a bifunctional enzyme encoded by gshF from Streptococcus thermophilus expressed in Escherichia coli. J. Biotechnol. 154, 261-268 (2011.

[2] Wang, D., Wang, C., Wu, H., Li, Z. & Ye, Q. Glutathione production by recombinant Escherichia coli expressing bifunctional glutathione synthetase. J. Ind. Microbiol. Biotechnol. 43, 45-53 (2016).

[3] Pophaly, S. D. et al. Glutathione biosynthesis and activity of dependent enzymes in food-grade lactic acid bacteria harbouring multidomain bifunctional fusion gene (gshF). J. Appl. Microbiol. 123, 194-203 (2017).

[4] Xiong, Z.-Q. et al. Functional analysis and heterologous expression of bifunctional glutathione synthetase from Lactobacillus. J. Dairy Sci.101 , 6937-6945 (2018).

[5] Cui, X. et al. Efficient glutathione production in metabolically engineered Escherichia coli strains using constitutive promoters. J. Biotechnol.289, 39-45 (2019).