Improved glutathione productionby gene expression in Escherichia coli

1. Key Laboratory of Industrial Biotechnology, Ministry of Education, Southern Yangtze University, Wuxi, China 2. Laboratory of Environmental Biotechnology, School of Biotechnology, Southern Yangtze University, Wuxi, China

Glutathione:

Glutathione (GSH, or L - γ -glutamyl-L-γ -cysteinylglycine)exists widely in nature and protects cells againstoxidation(Meister 1994). Its antioxidation function is mainly dueto its role in maintaining the normal redox environmentof cells (Izawa et al. 1995). GSH is now widely used inpharmaceutical, food and cosmetic industries. The com-mercial demand for GSH is expanding.

GSH is synthesized in two ATP-dependent steps:

L –glutamic acid + L –cysteine

γ -glutamylcysteine

synthetase gshI γ –glutamylcysteine

GSH synthetase gshII

GSH

Feedback inhibition

Aims:

To improve glutathione (GSH) production in Escherichia coli by different genetic constructions containing GSH genes.

Recombinant DNA methods

Standard recombinant DNA techniques were used as described by Sambrook et al. (1989).

Strains and plasmids

Escherichia coli JM109 was used as a host strain for cloning and fermentation experiments.

E. coli K12 was used as a source of chromosomal DNA for the cloning of gshI and gshII genes.

Plasmid pBV220 (an ampicillin-resistant and inducible expression vector, carrying the PRPL promoter)

gshI gene:

Forward primer : 5´-ATTTACCGcaattg ATGATCCCGGACGTATCAC-3 ´ Reverse primer : 5´ -AATTACCGcaattgTCAGGCGTGTTTTTCCAG-3´

caattg is Mun site

gshII gene:

Forward primer : 5 ´ -TTAACCGgtcgacAATTGCCATGACTGCCC-3´Reverse primer : 5´ -TTAACCGgtcgacTTACTGCTGCTGTAAACG-3 ´ gtcgac is SalI site

gshI b gene :

Forward primer : 5´ -ACCGAATTCctgcagGCATTCAAAGCAGAAGGC-3 ´Reverse primer : 5 ´- ACCGAATTCctgcagTCAGGCGTGTTTTTCCAG-3´

ctgcag is PstI site

Plasmid PBV01:

gshI gene was inserted into pBV220 plasmid treated with EcoRI to generate plasmid pBV01.

plasmid pBV02.

gshII gene was inserted into pBV01 plasmid treated with SalI togenerate plasmid pBV02.

plasmid pBV03.

gshI b gene was inserted into pBV02 plasmid treated with PstI to generate pBV03.

Protein analysis:

GSH production:

Conclusions: The simultaneous expression of two copies of gshI gene and one copy of gshII gene resulted in a significant increase in GSH production.

Significance and Impact of the Study: The expression strategy for GSH production described here can be used to increase gene expression and obtain high production rates in other multienzyme reaction systems.