Transcript Improved glutathione production by gene expression in

Improved glutathione production
by 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 due
to its role in maintaining the normal redox environment
of cells (Izawa et al. 1995). GSH is now widely used in
pharmaceutical, food and cosmetic industries. The commercial demand for GSH is expanding.
GSH is synthesized in two ATP-dependent steps:
L –glutamic acid + L –cysteine
γ -glutamylcysteine
synthetase
gshI
γ –glutamylcysteine
Feedback
inhibition
GSH synthetase
gshII
GSH
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 to
generate 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.