Bioprocess Biosyst Eng (2009) 32:729–735
DOI 10.1007/s00449-009-0297-x
ORIGINAL PAPER
Improved glutathione production by gene expression
in Pichia pastoris
Liwen Fei Æ Yan Wang Æ Shaoxin Chen
Received: 19 October 2008 / Accepted: 1 January 2009 / Published online: 20 January 2009
Ó Springer-Verlag 2009
Abstract To utilize Pichia pastoris to produce glutathi-
one, an intracellular expression vector harboring two genes
(gsh1 and gsh2) from Saccharomyces cerevisiae encoding
enzymes involved in glutathione synthesis and regulated by
the glyceraldehyde-3-phosphate dehydrogenase (GAP)
promoter was transformed into P. pastoris GS115. Through
Zeocin resistance and expression screening, a transformant
that had higher glutathione yield (217 mg/L) in flask cul-
ture than the host strain was obtained. In fed-batch culture
process, this recombinant strain displayed high activity
for converting precursor amino acids into glutathione. The
glutathione yield and biomass achieved 4.15 g/L and
98.15 g (dry cell weight, DCW)/L, respectively, after
50 h fermentation combined with addition of three amino
acids (15 mmol/L glutamic acid, 15 mmol/L cysteine, and
15 mmol/L glycine).
Keywords Glutathione production

Recombinant Pichia pastoris
Fed-batch culture
Introduction
Glutathione (GSH) is the most abundant non-protein thiol
found in all living organisms [1]. With its important
physiological functions such as antioxidant, detoxification
of xenobiotics and immune booster [2–8], GSH has been
widely used in medical treatment, food additive and cos-
metic industry [9, 10].
L. Fei
Y. Wang
S. Chen (&)
Department of Biochemistry,
Shanghai Institute of Pharmaceutical Industry,
Beijing Road West 1320, 200040 Shanghai,
People’s Republic of China
e-mail: sxzlb@263.net
In recent years, the demand for GSH is tending to
increase. However, the high price of GSH limits its
applications. Therefore, it is of great importance to develop
an efficient way to produce GSH. Compared with chemical
and enzymatic method, fermentative production of GSH is
an efficient approach in practical process [11]. To date,
Saccharomyces cerevisiae is the most commonly described
microorganism in literatures for GSH production. Many
attempts have focused on screening GSH over-producers
by mutagenesis and process optimization to achieve high
content of GSH [12–16]. Recently, Wang et al. reported a
strategy to increase the yield of GSH by amino acid
modulation and high-cell-density culture of S. cerevisiae
[12]. Although these processes have improved the pro-
duction of GSH, the volumetric yield of GSH was still low
because most of the strains belonging to S. cerevisiae were
hard to attain a very high-cell-density using minimal media
due to Crabtree effect [17].
Pichia pastoris is being widely used as a host organism
for the production of heterologous proteins [18]. The most
advantageous characteristic of this microorganism is that it
is able to grow to high biomass in simple minimal defined
media [above 130 g/L dry cell weight (DCW)] [19]. Since
P. pastoris has the ability to reach higher cell density than
that of S. cerevisiae during fermentation, the recombinant
P. pastoris has the potential to produce higher volumetric
yield of GSH.
In this work, a recombinant P. pastoris expressing the
genes of GSH synthesis enzymes, gsh1 and gsh2, was
constructed to increase GSH production. To our knowl-
edge, this is the first example of GSH production by
recombinant P. pastoris. This paper demonstrated that
the GSH production could be efficiently enhanced in
P. pastoris in fed-batch culture combined with addition of
precursor amino acids.
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730 Bioprocess Biosyst Eng (2009) 32:729–735

Materials and methods
Plasmids, strains, enzyme, and chemicals
All plasmids and strains used in this study are listed in
Table 1. Restriction endonuclease, T4 DNA ligase, and
DNA polymerase were obtained from TaKaRa (Dalian,
China). All other chemicals were of analysis grade and
commercially available.
Construction of integrative vectors
General recombinant DNA procedures were carried out
using standard procedure [20]. The construction flow chart
of integrative plasmid pGAPZHGSH is described in Fig. 1.
gsh1 (Gene ID. 853344) and gsh2 (Gene ID. 854108)
were amplified by PCR from genomic of S. cerevisiae. The
forward primer and reverse primer used for amplification
of gsh1 gene were 50 -CCATCGATACCATGGGACTCT
TAGCTTTG-30 (underlined sequence was ClaI site) and
50 -GCGGCCGCTTAACATTTGCTTTCTAT-30 (NotI site
was underlined), respectively. The obtained 2056-bp
product was inserted into pMD-18T to generate plasmid
pMDGSH1.
The forward primer and reverse primer used for ampli-
fication of gsh2 gene were 50 -CCATCGATACCATGGCA
CACTATCCA-30 (underlined was ClaI site) and 50 -GCG
G C C G C CCTAGTAAAGAATAATACTG-30 (underlined
was NotI site), respectively. The obtained 1496-bp PCR
product was inserted into pMD-18T, deriving plasmid
pMDGSH2.
his4 DNA fragment was amplified from plasmid
pAO815 with a forward primer 50 -GCGGATCCTAATGC
GGTAGTTTATCACAGTTA-30 (underlined was BamHI
site) and an antisense primer 50 -GAAGATCTTCATGACA
TTTCCCTTGCTACCTG-30 (underlined was BglII site).
The obtained PCR product was inserted into pMD-18T to
generate plasmid pMDHis4.
The 2921-bp BamHI-to-BglII fragment from plasmid
pMDHis4 was cloned into the BamHI site of pGAPZaA to
generate pGAPZaAH. Then, the 2034-bp ClaI-to-NotI
fragment containing gsh1 from pMDGSH1 was inserted
into the NotI-to-BstbI site of plasmid pGAPZaAH, obtaining
pGAPZHG1. Similarly, pGAPZG2 was constructed by
inserting ClaI-to-NotI fragment from pMDGSH2 into
BstbI-to-NotI site of pGAPZaA. Finally, the BglII-to-BamHI
fragment of pGAPZG2 containing GAP promoter, gsh2
and AOX1 terminator was subcloned into the BglII site of
pGAPZHG1, obtaining pGAPZHGSH.
Transformation and strain selection
Transformation of P. pastoris was achieved by electro-
poration according to the protocol of manufacture
(Invitrogen). The plasmid pGAPZHGSH linearized by
BspEI in the his4 DNA fragment was transformed into
P. pastoris GS115 by electroporation, and resulted trans-
formants were selected on yeast extract peptone dextrose
medium (YPD) plates containing 10 g/L yeast extract,
20 g/L peptone, 20 g/L glucose with addition of 1 mol/L
sorbitol and l00 lg/mL Zeocin.

Table 1 Plasmids and strains used in this study

Plasmids Relevant characteristics Source or reference

pMD-18T Amp resistant TaKaRa

pGAPZaA Zeocin resistance, pGAP, a-factor, AOX1 terminator Invitrogen
pAO815 G418 resistant, pAOX1, his4 Invitrogen
pMDGSH1 gsh1 in pMD-18T, Amp resistant This work
pMDGSH2 gsh2 in pMD-18T, Amp resistant This work
pMDHis4 his4 in pMD-18T, Amp resistant This work
pGAPZaAH his4 in pGAPZaA, Zeocin resistance, pGAP, a-factor, AOX1 terminator This work
pGAPZHG1 gsh1 in pGAPZaAH, Zeocin resistance, his4, pGAP, AOX1 terminator This work
pGAPZG2 gsh2 in pGAPZaA, Zeocin resistance, pGAP, AOX1 terminator This work
pGAPZHGSH gsh1 and gsh2 in pGAPZaA, Zeocin resistance, his4, pGAP, AOX1 terminator This work
Strains
E.coli DH5a F-, SupE44, lacU169, a80/lacZDM15, deoR, hsdR17, phoA, recA1, endA1, gyrA96, k-, thi-1, relA1 Amersham
P. pastoris GS115 his4-, Mut? Invitrogen
P. pastoris D18 gsh1 and gsh2 integrated into P. pastoris GS115 This work
S. cerevisiae Wild type strain containing the gsh1 and gsh2 gene Our laboratory

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Bioprocess Biosyst Eng (2009) 32:729–735 731

Fig. 1 Construction of AOX1 promoter fragment

integrative plasmid
pGAPZHGSH. pGAPZaAH AOX1 TT
AMPResist
was obtained by inserting his4 AMP Bam HI
pBR322ori
fragment from pAO815 to
pAO815
BamHI site of pGAPZaA; gsh1
7709 bp
was inserted into pGAPZaAH to
generate pGAPZHG1; gsh2 was
3-AOX1
inserted into pGAPZaA to his4 ORF
generate pGAPZG2;
pGAPZHGSH was constructed Bgl II pGAP
to express the cassette of genes Bst BI Multi cloning site
Bgl II Not I
pGAP
of gsh1 and gsh2 under control a-factor signal
of GAP promoter. Zeocin was AOX1 TT
Bst BI his4 Zeocin
used for positive colony a-factor signal Multi cloning site
Bam HI
pGAPZαAH
selection 6063 bp
pGAPZαA
3147 bp Not I

AOX1 TT Bsp EI
his4
Zeocin Bam HI his4 fragment from pAO815

gsh2 gsh1
Bgl II
pGAP
Bgl II pGAP
Bam HI
Zeocin

gsh1
pGAPZG2
4308 bp gsh2 pGAPZHG1
Zeocin 7784 bp

Bsp EI
AOX1 TT
Bam HI Bam HI
AOX1 TT his4

Bgl II pGAP

Zeocin gsh2

AOX1 TT
Bsp EI pGAPZHGSH pGAP
10172 bp

his4

gsh1

AOX1 TT

Each colony growing on the plate was picked into a
250 mL shake flask containing 15 mL YPD medium and
cultured at 30 °C and 250 rpm for 48 h. 1 mL of each
culture was centrifuged at 4,0009g for 5 min and the
collected cells were washed twice with deionized water
before resuspended in 1 mL deionized water. Intracellular
GSH was extracted from the cells by freezing the resus-
pended cells at -20 °C overnight, and then incubating at
83 °C for 15 min. After centrifugation, the resultant
supernatant was used for assay of GSH content by colori-
metric method described previously [21, 22]. The posi-
tive colonies were subjected to PCR for confirmation of
the presence of gsh1 and gsh2. The sense primer was
50 -GGTCTGACGCTCAGTGGAACGAAAC-30 , and the
antisence primer was 50 -GATTCGATAGATCCACAGGA
CGGGTG-30 .
To examine the genetic stability of the recombinant
strain, the colony on YPD agar plate was picked to a YPD
agar slant and cultivated at 30 °C for 48 h. Biomass formed
on this slant was called generation F0. Generation F1–F5
slant were produced by inoculating the biomass from their
parent slant followed with 48 h cultivation at 30 °C. Cells
on each generation of slant were separately inoculated to a
250 mL shake flask containing 15 mL YPD medium and

123
732
cultured at 30 °C for 18 h. Ten percent (v/v) of the culture
was transferred into 15 mL of fresh YPD medium for
cultivation of 48 h, and the GSH content was analyzed as
described above.
Medium optimization
Various carbon and nitrogen sources were tested to deter-
mine the best components for GSH production. P. pastoris
D18 growing on YPD agar slant was cultured in liquid
YPD medium at 30 °C and 250 rpm for 18 h as seed cul-
ture. For all experiments, 10% inoculum (v/v) of seed
culture was used in a 500 mL shake flask containing
50 mL culture medium and cultivated at 30 °C and
250 rpm for 48 h. The GSH content was analyzed as
described above.
Fed-batch fermentation
Fed-batch fermentation was carried out in a 5 L fermentor
(B. Braun, Biostat B). Strain P. pastoris D18 grown on
YPD agar plate was inoculated into liquid YPD medium
and cultured at 30 °C until OD600 value reached 5.0, and
then was used as seed culture. Ten percent inoculum (v/v)
was transferred into the bioreactor containing 50 g/L
glucose, 5 g/L yeast extract, 11 g/L (NH4)2SO4, 7 g/L
K2HPO4
3H2O, 5 g/L MgSO4
7H2O, 0.5 g/L CaCl2
2H2O,
0.5 g/L KCl with pH 5.0–5.5. Initial agitation was 250 rpm
and the temperature was 30 °C. Dilute oxygen (DO) was
maintained above 25% through adjustment of agitation
speed. Air was used and the aeration rate in the fed-batch
culture was 1 vvm. The pH of the medium was adjusted to
5.0 with 10% ammonium hydroxide. Feeding of 80% (w/v)
glucose was carried out after 16 h of culture at a rate of
2.9 mL/L h by peristaltic pump. Dry cell weight was
determined after yeast cells were centrifuged at 4,0009g
for 10 min and washed twice with deionized water before
drying at 105 °C to constant weight, and the GSH con-
centration was analyzed by HPLC.
Analytical method
Glutathione was extracted from the fermentation broth by
adjusting the pH of samples to 1.5 with 2.0% (v/v) H2SO4
and incubated at 85 °C for 5 min. And then GSH con-
taining supernatant was centrifuged at 4,0009g for 5 min
and filtered through 0.45 lm membrane before subjected to
HPLC analysis. The column used was C18 (Hypersil BDS,
150 9 4.6 mm, 5 lm). The mobile phase consisted of
methanol and 0.11% (w/v) 1-heptanesulfonate (sodium
salt) in potassium phosphate buffer (50 mmol/L, pH 3.0) in
the ratio of 5:95 (v/v). The flow rate was 1.0 mL/min, and
123
Bioprocess Biosyst Eng (2009) 32:729–735
detection wavelength was set at 210 nm. GSH from Koway
(Japan) was used as calibration standard.
Results and discussion
Construction and selection of recombinant P. pastoris
The biosynthesis pathway of GSH has been elucidated,
which includes a two-step enzymatic catalyzed process
[23–25]. First, gamma-glutamylcysteine is synthesized
from L-glutamic acid and L-cysteine via gamma-glutamyl-
cysteine synthetase (GSH1); second, glycine is added to the
C-terminal of gamma-glutamylcysteine by the GSH syn-
thetase (GSH2). And the DNA sequences of the two
enzymes have been identified in S. cerevisiae [26, 27].
Therefore, an integrative plasmid pGAPZHGSH was con-
structed in order to obtain a recombinant P. pastoris for
GSH synthesis (Fig. 1). The plasmid contains a strong
constitutive GAP promoter, the his4 DNA fragment for
homologous recombination into the his4 locus on the
chromosome of P. pastoris, the gsh1 and gsh2, the AOX1
terminator, and the Zeocin-resistant gene. In addition, the
a-signal factor was deleted from the pGAPZaA, so that the
enzymes could be expressed intracellularly in P. pastoris.
pGAPZHGSH was linearized by BspEI and transformed
into P. pastoris GS115. Transformants that appeared on the
YPD plate containing 1 mol/L sorbitol and 100 lg/mL
Zeocin after 48–72 h revealed that plasmid had been
integrated into the yeast chromosome through homologous
recombination. And the presence of the gsh1 and gsh2 in
recombinant P. pastoris was also confirmed by PCR using
the primers described in the ‘‘Materials and methods’’. The
copy number of the integrated plasmid may vary in dif-
ferent transformants resulting in diverse expression levels
of cloned gene. Three hundred colonies were picked to
assess the GSH-producing capability. As expected, the
GSH levels varied from different colonies transformed by
the same linearized plasmid. As shown in Fig. 2, the GSH
content ranged from 35 to 230 mg/L, and approximately
10% of the strains produced GSH higher than 200 mg/L.
By isolation and purification for several times, the one
colony originally designated as P. pastoris D18, which
produced 217 mg/L GSH in the flask culture, 4.7 times
higher than that of host strain (38 mg/L), was selected for
further study. The genetic stability of the recombinant
strain was also taken into consideration by analyzing the
GSH-production level. It was observed that the GSH-pro-
duction capability showed no obvious change after serial
cultivation for five generations, suggesting that the plasmid
pGAPZHGSH was stably integrated into the chromosome
of P. pastoris.
Bioprocess Biosyst Eng (2009) 32:729–735 733

10%
15%
16%
59%
0-50(mg/L) 51-100(mg/L)
101-200(mg/L) 201-250(mg/L)
Fig. 2 Preliminary screening of GSH-producing recombinant P.
pastoris in flask culture. Transformants growing on the Zeocin-resistant
selection plates were picked and cultured in 15 ml of YPD liquid
medium at 30 °C for 48 h. GSH content was analyzed by colorimetric
method
Fed-batch culture
In order to enhance the synthesis level of GSH by P. pas-
toris D18, the composition of the production medium was
examined before batch culture. Several different carbon
sources and nitrogen sources were investigated (Table 2),
and the glucose and yeast extract were found to be the best
carbon source and nitrogen source for GSH synthesis,
respectively. Finally, a medium described in the part of
materials and methods was applied for fed-batch culture.
To scale up the fermentation of P. pastoris D18, the
batch fermentation for GSH production was carried out in
a 5 L fermentor. The time course of fed-batch culture of
P. pastoris D18 was shown in Fig. 3. During the process of
fermentation, the cell density rose continually, and grew up
to 94.98 g (DCW)/L at 45 h. The GSH content reached
0.92 g/L at 28 h and tended to remain constant during
further culture. Unexpectedly, as a biomass related product,
the result showed that the GSH content did not increase
with the accumulation of biomass after 28 h.
It has been reported that GSH yield could be enhanced
effectively by addition of precursor amino acids mixture
[28–30]. Therefore, the effect of concentrations of pre-
cursor amino acids on GSH production was investigated in
fed-batch culture of strain P. pastoris D18. As shown in
Table 3, the addition of amino acids significantly stimu-
lated the GSH synthesis in the yeast cells, and the GSH
yield and productivity were increased with increasing the
concentration of each amino acid at 0–15 mmol/L. How-
ever, in the case of 20 mmol/L amino acid, there was little
increase of yield and productivity. In addition, we have
observed that GSH yield was not only influenced by the
concentration of amino acids but also related to the time
that amino acids were added. When the amino acids were
added at culture time earlier than 30 h, the effect of amino
acids on GSH production was inconspicuous. The optimal
feeding time was found to be 40–45 h when P. pastoris
D18 biomass reached above 90 g (DCW)/L.
The time course of fed-batch cultivation of P. pastoris
D18 with amino acids addition was shown in Fig. 4. When
15 mmol/L of each amino acid was added at 44 h, the GSH
content was increased rapidly from 1.03 to 4.15 g/L at
50 h, which was 351% higher than that without any amino
acid addition. The biomass reached 98.15 g (DCW)/L at
1.5 120

Table 2 Effects of carbon sources and nitrogen sources on GSH

GSH production (g/L)

Dry cell weight (g/L)

production 90
a b 1.0
Carbon source GSH yield Nitrogen source GSH yield
(mg/L) (mg/L)
60

Glucose 227.9 Yeast extract 285.4

0.5
Glycerol 217.8 Peptone 265.9
30
Sucrose 159.2 Corn steep liquor powder 208.9
Corn gluten meal 152.5
0.0 0
P. pastoris D18 cultured in the YPD medium in which GSH yield was 0 10 20 30 40 50
227.9 mg/L was used as the control. Each trial was conducted in Culture time (h)
triplicate
a GSH production Dry cell weight
2% (w/v) of various carbon sources were instead of glucose in the
YPD medium Fig. 3 Fed-batch culture of P. pastoris D18 without amino acids
b
3% (w/v) nitrogen sources were instead of yeast extract and peptone addition. Fermentation was carried out in a 5 L fermentor at 30 °C
in the YPD medium and pH 5.0. Feeding of 80% (w/v) glucose was carried out at 16 h

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734 Bioprocess Biosyst Eng (2009) 32:729–735

Table 3 Effect of precursor amino acids on synthesis of glutathione in fed-batch culture of P. pastoris D18
Amino acids mixture GSH yield GSH productivity (mg/L
h)
(g/L)

Without addition of amino acid 0.92 32.8

5 mmol/L glutamic acid, 5 mmol/L cysteine, 5 mmol/L glycine 2.03 40.6
10 mmol/L glutamic acid, 10 mmol/L cysteine, 10 mmol/L glycine 2.72 54.4
15 mmol/L glutamic acid, 15 mmol/L cysteine, 15 mmol/L glycine 4.15 83.0
20 mmol/L glutamic acid, 20 mmol/L cysteine, 20 mmol/L glycine 4.26 83.5
Fed-batch culture was carried out in a 5 L fermentor at 30 °C and pH 5.0. The fermentation time was 50 h, and amino acids were added at 44 h

5.0 120 course was controlled within 50 h. All the factors con-
tribute to low production cost and therefore make it
possible to produce GSH in industrial scale.
GSH production (g/L)

4.0

Dry cell weight (g/L)

90

3.0
Conclusion
60
2.0
The present studies show that the recombinant P. pastoris
30 expressing heterologous GSH synthesis genes is an alter-
1.0
native microorganism for GSH production. The high-cell-
density culture and amino acid addition were successfully
0.0 0 combined together to maximize GSH production by cul-
0 10 20 30 40 50 60
Culture time (h) turing the recombinant P. pastoris, which allows to obtain
GSH production Dry cell weight
GSH with high yield and relatively low cost.

Fig. 4 Fed-batch culture of P. pastoris D18 with amino acids Acknowledgments This work was supported by Creative Foundation
addition. Fermentation was carried out in a 5 L fermentor at 30 °C Grant of Shanghai Institute of Pharmaceutical Industry.
and pH 5.0. Feeding of 80% (w/v) glucose was carried out at 16 h.
Three amino acids mixture (15 mmol/L glutamic acid, 15 mmol/L
cysteine and 15 mmol/L glycine) were added at 44 h
References

the end of fermentation. The result indicated that the 1. Meister A, Andersen ME (1983) Glutathione. Annu Rev Biochem
52:711–760
recombinant yeast displayed high GSH synthesis activity 2. Flohe L (1985) The glutathione peroxidase reaction: molecular
for converting the three precursor amino acids into GSH in basis of the antioxidant function of selenium in mammals. Curr
high-cell-density culture process. During the fermentation Top Cell Regul 27:473–478
process, the glucose was fed to obtain a high-density of 3. Vartanyan LS, Gurevich SM, Kozachenko AI, Nagler LG,
Lozovskaya EL, Burlakova EB (2000) Changes in superoxide
biomass. The total consumption of glucose (including the production rate and in superoxide dismutase and glutathione
glucose used in fermentation medium) was 130 g/L of peroxidase activities in subcellular organelles in mouse liver
fermentation broth, and the GSH yield on glucose was under exposure to low doses of low-intensity radiation. Biochem
32 mg (GSH)/g (glucose). (Mosc) 65:442–446
4. Ray S, Watkins DN, Misso NL, Thompson PJ (2002) Oxidant
Recently, production of GSH by S. cerevisiae or Can- stress induces gamma-glutamylcysteine synthetase and glutathi-
dida utilis were reported by several research groups, the one synthesis in human bronchial epithelial NCI-H292 cells. Clin
reported yield of GSH in batch culture ranged from 800 to Exp Allergy 32:571–577
2,000 mg/L [12–14, 16]. Among them, Wen et al. obtained 5. Singh RJ (2002) Glutathione: a marker and antioxidant for aging.
J Lab Clin Med 140:380–381
the maximal GSH yield (2.19 g/L) by high-cell-density 6. Rolseth V, Djurhuus R, Svardal AM (2002) Additive toxicity of
fed-batch culture of S. cerevisiae and amino acid modu- limonene and 50% oxygen and the role of glutathione in detox-
lation [14]. In our study, the recombinant P. pastoris D18 ification in human lung cells. Toxicology 170:75–88
showed higher biomass and GSH synthesis capability 7. Penninckx M (2000) A short review on the role of glutathione in
the response of yeasts to nutritional, environmental, and oxidative
during fed-batch culture. Moreover, glucose was used as stresses. Enzyme Microb Technol 26:737–742
carbon source instead of glycerol, which is a common 8. Droge W, Breitkreutz R (2000) Glutathione and immune func-
carbon source for P. pastoris fermentation, and the time tion. Proc Nutr Soc 59:595–600

123
Bioprocess Biosyst Eng (2009) 32:729–735
9. Wei G, Li Y, Du G, Chen J (2003) Effect of surfactants on
extracellular accumulation of glutathione by Saccharomyces
cerevisiae. Process Biochem 38:1133–1138
10. Yoshida K, Hariki T, Inoue H, Nakamura T (2002) External skin
preparation for whitening. JP Patent 2,002,284,664
11. Sakato K, Tanaka H (1992) Advanced control of glutathione
fermentation process. Biotechnol Bioeng 40:904–912
12. Wang Z, Tan T, Song J (2007) Effect of amino acids addition and
feedback control strategies on the high-cell-density cultivation of
Saccharomyces cerevisiae for glutathione production. Process
Biochem 42:108–111
13. Zhang T, Wen S, Tan T (2007) Optimization of the medium for
glutathione production in Saccharomyces cerevisiae. Process
Biochem 42:454–458
14. Wen S, Zhang T, Tan T (2006) Maximizing production of
glutathione by amino acid modulation and high-cell-density
fed-batch culture of Saccharomyces cerevisiae. Process Biochem
41:2424–2428
15. Kazue Y, Kaori Y (2006) Mutant yeast, method of producing
glutathione-rich yeast, culture thereof, fraction thereof, yeast
extract and glutathione-containing foods and drinks. WO Patent
2,006,013,736
16. Santos LO, Gonzales TA, Ubeda BT, Alegre RM (2007) Influ-
ence of culture conditions on glutathione production by
Saccharomyces cerevisiae. Appl Microbiol Biotechnol 77:763–
769
17. Leuenberger HG (1972) Cultivation of Saccharomyces cerevisiae
in continuous culture II. Influence of the crabtree effect on the
growth characteristics of Saccharomyces cerevisiae grown in a
glucose limited chemostat. Arch Mikrobiol 83:347–358
18. Cereghino JL, Cregg JM (2000) Heterologous protein expression
in the methylotrophic yeast Pichia pastoris. FEMS Microbiol
Rev 24:45–66
735
19. Shay LK, Hunt HR, Wegner GH (1987) High productivity fer-
mentation process for cultivating industrial microorganisms. J Ind
Microbiol 2:79–85
20. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory
manual, 3rd edn edn. Cold Spring Harbor Laboratory Press, New
York
21. Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem
Biophys 82:70–77
22. Liu J, Wang Y-q, Liu G, Zhang B-r (2004) Comparison of three
methods for determination of glutathione (Chinese). J Beijing
Univ Chem Technol 31:35–38
23. Johnston RB, Bloch K (1951) Enzymatic synthesis of glutathione.
J Biol Chem 188:221–240
24. Snoke JE, Bloch K (1952) Formation and utilization of gamma-
glutamylcysteine in glutathione synthesis. J Biol Chem 199:407–
414
25. Snoke JE, Yanari S, Bloch K (1953) Synthesis of glutathione
from gamma-glutamylcysteine. J Biol Chem 201:573–586
26. Ohtake Y, Yabuuchi S (1991) Molecular cloning of the gamma-
glutamylcysteine synthetase gene of Saccharomyces cerevisiae.
Yeast 7:953–961
27. Inoue Y, Sugiyama K, Izawa S, Kimura A (1998) Molecular
identification of glutathione synthetase (GSH2) gene from Sac-
charomyces cerevisiae. Biochim Biophys Acta 1395:315–320
28. Alfafara C, Kanda A, Shioi T, Shimizu H, Shioya S, Suga K
(1992) Effect of amino acids on glutathione production by Sac-
charomyces cerevisiae. Appl Microbiol Biotechnol 36:538–540
29. Cha JY, Park JC, Jeon BS, Lee YC, Cho YS (2004) Optimal
fermentation conditions for enhanced glutathione production by
Saccharomyces cerevisiae FF-8. J Microbiol 42:51–55
30. Wen S, Zhang T, Tan T (2005) Optimization of the amino acid
composition in glutathione fermentation. Process Biochem
40:3474–3479
123