chenjian-15.pdf

7/27/2019 chenjian-15.pdf

1/10

MIN I-REV IEW

Yin Li . Gongyuan Wei . Jian Chen

Glutathione: a review on biotechnological production

Received: 1 June 2004 / Revised: 16 August 2004 / Accepted: 31 August 2004 / Published online: 12 October 2004# Springer-Verlag 2004

Abstract This Mini-Review summarizes the historicdevelopments and technological achievements in the

biotechnological production of glutathione in the past 30

years. Glutathione is the most abundant non-protein thiolcompound present in living organisms. It is used as a

pharmaceutical compound and can be used in foodadditives and the cosmetic industries. Glutathione can be

produced using enzymatic methods in the presence of ATPand its three precursor amino acids (L-glutamic acid, L-cysteine, glycine). Alternatively, glutathione can be

produced by direct fermentative methods using sugar asa starting material. In the latter method, Saccharomycescerevisiae and Candida utilis are currently used to produceglutathione on an industrial scale. At the molecular level,the genes gshA and gshB, which encode the enzymes -glutamylcysteine synthetase and glutathione synthetase,

respectively, have been cloned from Escherichia coli andover-expressed in E. coli, S. cerevisiae, and Lactococcuslactis. It is anticipated that, with the design and/ordiscovery of novel producers, the biotechnological

production of glutathione will be further improved toexpand the application range of this physiologically andmedically important tripeptide.

Introduction

Glutathione (-glutamyl-L-cysteinylglycine, GSH) is themost abundant non-protein thiol compound widely

distributed in living organisms and, predominantly, ineukaryotic cells (Meister and Anderson 1983). While over90% of the glutathione is normally present in the reduced

form GSH, several additional forms of glutathione arepresent in (microbial) cells, tissues, and plasmas. Gluta-thione disulfide GSSG (oxidized glutathione), formedupon oxidation of GSH, can be in turn be reduced to GSH

by glutathione reductase at the expense of NADPH(Carmel-Harel and Storz 2000). Besides GSSG, GSHmay occur in other forms of mixed disulfides, for example,GS-S-CoA, GS-S-Cys (Penninckx 2002), and GS-S-pro-tein which is formed via glutathionylation.

Although GSH has been found to be involved in manyphysiological processes and to play various importantroles, the major and general functions of GSH can besummarized into three major ways, i.e. serving as

antioxidant, immunity booster, and detoxifier in highereukaryotic organisms (Pastore et al. 2003). First, thestrong electron-donating capability of GSH and therelatively high intracellular concentration (up to millimolarlevels) enable the maintenance of a reducing cellularenvironment. This makes GSH an important antioxidantfor protecting DNA, proteins, and other biomoleculesagainst oxidative damage generated by, for example,reactive oxygen species. Second, GSH plays an importantrole in immune function via white blood cell productionand is one of the most potent anti-viral agents known.Finally, GSH can be conjugated to exogenous electro-

philes and diverse xenobiotics by glutathione-S-transfer-

ase to accomplish detoxification. GSH is thus consideredto be one of the most powerful, versatile, and importantself-generated defense molecules. In humans, GSH defi-ciency has been linked to a number of disease states: HIVinfection, liver cirrhosis, pulmonary diseases, gastrointes-tinal and pancreatic inflammations, diabetes, neurodegen-erative diseases, and aging (Wu et al. 2004). GSH iswidely used as a pharmaceutical compound and has the

potential to be used in food additives and in the cosmeticindustries (Sies 1999), given that the price can be furtherdecreased by improving production methods.

Y. Li (*) . G. Wei . J. ChenThe Key Laboratory of Industrial Biotechnology, Ministry ofEducation; School of Biotechnology, Southern YangtzeUniversity,170 Huihe Road,Wuxi, 214036, Peoples Republic of Chinae-mail: [email protected].: +86-510-5885727Fax: +86-510-5888301

G. WeiSchool of Life Science, Soochow University,Suzhou, 215007, Peoples Republic of China

http://www.paper.edu.cn


2/10

While the physiological roles of GSH in human andanimal tissues, in plant cells, and in microbial cells have

been extensively explored and described (Carmel-Hareland Storz 2000; Penninckx and Elskens 1993; Penninckx2000, 2002), reviews covering the biotechnological

production of this medically important tripeptide arescarce. One reason might be that much of the outcomeof research on the production of GSH is patented, due to

the great economic interest. The latest reviews describingthe production of GSH were published nearly 10 years ago(Murata 1989, 1994; Murata and Kimura 1990), withspecial emphasis on the production of GSH by geneticallyengineered Escherichia coli or Saccharomyces cerevisiae.

In this Mini-Review, we aim to provide a summary ofthe historic developments and technological achievementsin the biotechnological production of GSH in the past 30years. Patents relevant to enzymatic and fermentative

production of GSH are cited, to show the diversity ofglutathione-producing approaches. Furthermore, future

perspectives on the biotechnological production of gluta-thione are given.

Production of glutathione: a brief historic overview

GSH was discovered in ethanol-extract of bakers yeast in1888 and was referred to as philothion. It wassubsequently renamed glutathione follow the establish-ment of its molecular structure in 1921 (Penninckx andElskens 1993). With the discovery of the presence of GSHin many living organisms, solvent extraction of GSH fromanimal or plant tissues was exploited as a preparativeapproach. However, the limited raw materials availableand the relatively low intracellular content of GSH made

the end-product expensive, thus hampering its practicalapplication.

Nearly 70 years ago, it was demonstrated that glutathi-one could be synthesized by a chemical method(Harington and Mead 1935). The introduction andremoval of sulfhydryl protective agents on a cysteineresidue were crucial steps in the chemical synthesis ofGSH (Douglas 1989). Although this process was com-

mercialized in the 1950s, chemically synthesized GSHwas an optically inactive (racemic) mixture of the D- and L-isomers. As only the L-form is physiologically active, anoptical resolution is required to separate the L-form fromits D-isomer.

Following the observation of the biosynthesis of GSH inan isolated liver and the characterization of the biosyn-thetic pathway for GSH (Bloch 1949), explorations on theenzymatic and fermentative production of GSH were

pursued. GSH is synthesized in two consecutive ATP-dependent reactions (Meister and Anderson 1983). Thedipeptide -glutamylcysteine (-GC) is first synthesizedfrom L-glutamic acid and L-cysteine by -glutamylcysteine

synthetase (also known as glutamate-cysteine ligase, EC6.3.2.2, GSHI). In the second step, catalyzed by glutathi-one synthetase (EC 6.3.2.3, GSHII), glycine is added tothe C-terminal site of-GC to form GSH. Generally, theactivity of GSHI is feedback-inhibited by GSH (but notGSSG) to avoid over-accumulation of GSH, which is of

physiological significance (Richman and Meister 1975).Meanwhile, cellular GSH can be degraded by -

Fig. 1 Overview of GSH-related metabolism pathways and thegeneral physiological roles played by GSH in microorganisms. Cyscysteine, Gly glycine, Glu glutamate, G6PDH glucose-6-phosphatedehydrogenase, GPx glutathione peroxidase, GR glutathione reduc-tase, GRX glutaredoxin, GSHI -glutamylcysteine synthetase,GSHII glutathione synthetase, GTP -glutamyltransferase, ROOHperoxides, ROH reduced peroxides, SS disulfide bond

Table 1 Development of thebiotechnological production ofGSH

Research interest in GSH production Period

Breeding of yeast (predominantly S. cerevisiae and Candida utilis) for glutathione

production, using either fermentative or enzymatic methods

19761985,

1998present

Purification of glutathione from biological systems 19761987,19931998

Selection of bacteria to produce glutathione using enzymatic methods 19781991

Construction of ATP regeneration systems 19781982

Molecular biology of GSH biosynthesis in E. coli and construction of recombinant

E. coli to produce GSH

19821990

Construction of recombinant S. cerevisiae to produce GSH 19861990,

19961998

Optim ization and control of glutathione production process 19911994,

1997present

Production of glutathione by lactic acid bacteria 2001present

234 http://www.paper.edu.cn


3/10

Table2

Anoverviewoftheliteratureontheenzymaticproductionofglutathionebymicroorganisms.Enzymaticproductionofglutathioneusesareactionmix

turecontainingL-glutamic

acid,L-cysteine,andglycine

GSHbiosynthesis

ATPregeneration

ATPsupply

T (C)

pH

t (h)

GSH

(mgl1)

Yieldon

Cys(%)

Reference

C.

kruseiIFO0011

Notnecessary

37

6.0

16

350

1.4

Yokozekietal.

(1985)

S.cerevisiaeIFO2044(immobilize

d)

Glycolysis

(glucose)

30

7.5

5

240

7.8

Chibataetal.

(1979b)

S.cerevisiaeIFO0304(treatedbysodiumlaurylsulfate)

Glycolysis

(glucose)

30

7.5

18

11.1

24.1

Takesueetal.

(1979)

S.cerevisiaeIFO0021(treatedbysodiumdodecylsulfateand

-1,3-glucanase)

Glycolysis

(glucose)

37

7.3

15

9,060

98.4

Miwa(1978)

S.glyoxalphilusCDA-5(treatedby

sodiumdodecylsulfate,

immobilized)

Glycolysis

(glucose)

37

7.3

Continuous8,989

97.6

MiwaandTajima

(1978a)

S.cerevisiae500(immobilizedcrudeenzymesofGSHI,

GSHII)

Immobilizedcarbamyl-

phosphokinase

fromStreptococcus

faecalisR600

ATP

35

7.3

5

325

58.6

Miwa(1976)

S.cerevisiae(immobilizedGSHI,G

SHII)

ATP

30

7.5

2,517

82

MiyamotoandMiwa

(1977)

AchromobacterlacticumFERM-P7401

Notnecessary

37

6.0

16

560

2.2

Yokozekietal.

(1985)

CorynebacteriumglutamicumATCC21171(frozencells

treatedwithsurfactant,xylene)

Brevibacterium

ammoniagenes

ATCC21170

(frozen

cellstreated

withsurfactan

t,

xylene)

Glycolysis

(glucose)

37

7.4

6

2,456

49.0

KyowaHakkoKogyo

Co.(1985b)

E.

coli(treatedwithsurfactant,xylene)

ATP

37

7.4

6

2,714

58.9

KyowaHakkoKogyo

Co.(1985a)

E.

coliATCC11303

Glycolysis

(glucose)

37

7.4

2,700

35.2

Fujioetal.

(1985)

E.

coliBATCC23226(crudeenzy

mesofGSHI,GSHII)

Acetatekinase

preparedfrom

E.

coliBATC

C23226

Dextran-N-[(6-ami-

nohexyl)-

carbamoylmethyl]

ATP

37

7.0

3

46.3

3.0

Chibataetal.

(1979a)

Proteusmirabilis(cell-freeextract)

Driedyeast

ATP,glucose

37

8.5

2

1,320

61.4

AjinomotoCo.(1984)

P.mirabilisIFO3849

ATP

37

9.1512

280

1.3

AjinomotoCo.(1982a)

P.vulgaris(crudeGSHI,GSHIIenzymesmodifiedbyN-

ethylmaleimide

ATP

37

9.1512

480

AjinomotoCo.(1982b)

Phormidiumlapideum(2,5

00lx)

Lightusedasan

externalenerg

ysource

ATP

47

7.5

12

1.5mgg

1

wetcell

Sawaetal.

(1986)

235



4/10

glutamyltranspeptidase (-GTP) to transfer the -glutamylmoiety (Meister and Anderson 1983), which is importantin amino acid transport. To overproduce GSH using eitherenzyme/intact cell biocatalysis or fermentation, the releaseof GSHI from feedback inhibition by GSH and/or theinactivation or deficiency of-GTP are necessary (Murata1994). Figure 1 gives a biochemical overview, showingthe biosynthetic and metabolic pathways and illustrating

the general physiological roles of GSH.Table 1 summarizes the development of the biotechno-

logical production of GSH. Research on fermentative andenzymatic production of GSH was very active between1976 and 1985 in Japan; and fermentative production ofGSH by yeast was commercialized in the early 1980s.Since then, the number of patents on the production ofGSH declined dramatically. To date, the enzymatic

production of GSH has not been commercialized becauseof the relatively high production cost.

Enzymatic production of glutathione

One of the well studied approaches to glutathioneproduction is the enzymatic method. In principle, theessential elements to constitute an enzymatic synthesissystem include: GSHI, GSHII, precursor amino acids (L-glutamic acid, L-cysteine, glycine), ATP, necessarycofactors (Mg2+) to maintain the activities of GSHI andGSHII, and a suitable pH (usually pH 7.5). Table 2 givesan overview of the enzymatic production of GSH usingeither intact cells or crude enzymes. It can be generallyconcluded from Table 2 that the addition of ATP is notnecessary if using S. cerevisiae, but is required if using

prokaryotes or crude enzymes. This is due to the fact that,

in S. cerevisiae, the strong ATP regeneration capability ofglycolysis compensates the consumption of ATP. Also,increasing the cell wall permeability (induced by freezingthawing and/or treatment with surfactants or enzymes)enhances enzymatic production significantly when intactcells are used.

The requirement for ATP in the enzymatic production ofglutathione makes this process difficult to scale-up, since itis impractical from an economic point of view to add ATPdirectly on an industrial scale. Accordingly, it would be ofindustrial interest to construct a highly efficient ATPregeneration system, which can be briefly defined as asystem in which ATP-requiring reactions are coupled with

ATP-producing reactions. ATP regeneration systems canbe categorized into self-coupling systems which workwithin one organism and co-coupling systems which areconstructed between two or more organisms. When a co-coupling ATP regeneration system is used, the efficiencyof ATP/ADP transport across the respective cell mem-

branes should be carefully taken into account.Self-coupling ATP regeneration systems have not been

intensively investigated, since it is difficult to simulta-neously improve the activities of both GSH biosynthesisand ATP regeneration in one organism (Shimosaka et al.1982). The co-coupling ATP regeneration system is the

only feasible approach to date. The reaction catalyzed byacetate kinase in E. coli cells was explored as an ATPgeneration system to be coupled with GSH biosynthesisreactions, resulting in the successful production of GSH(Langer et al. 1976). However, the acetyl phosphatesubstrate required for this approach was unstable andexpensive. Murata and co-workers considered the glyco-lytic pathway of S. cerevisiae to be the simplest and the

most capable system for regenerating sufficient ATP forGSH biosynthesis (Murata et al. 1981b). When theeconomic supply of ATP was no longer a problem, thelow activities of GSHI and GSHII became the limitingfactors in GSH biosynthesis. The requirement to enhancethe activities of GSHI and GSHII accelerated theapplication of molecular cloning and genetic engineeringapproaches in GSH biosynthesis.

Fermentative production of glutathione

GSH producer screening

The advantage of the enzymatic production of GSH is thata high concentration (up to 9 g l1; Table 2) can beachieved. However, since the usage of three precursoramino acids increases the production cost, the fermentative

production of glutathione using sugar materials assubstrates has been extensively studied and is the majorcommercial method currently used. S. cerevisiae andCandida utilis are the most commonly used microorgan-isms on an industrial scale; and the GSH contents of thewild-type strains are usually high (0.11.0% dry cellweight). Therefore, these two microorganisms werechosen as targets for mutagenesis. The physical or

chemical mutagenesis methods used included UV, X-radiation, -radiation, and N-methyl-N-nitro-N-nitroso-guanidine (NTG) treatment, while resistance to com-

pounds such as glutathione analogues (methionine, ethi-onine), 1,2,4-triazole, sodium cyanide, and sulfite wasused to screen GSH over-producers. The mechanism forthe selection, in most cases, was to disrupt or release thefeedback inhibition of GSH on GSHI. Several examplesillustrate how powerful the screening strategy was: (1) C.utilis n74-8 sensitive to DL-ethionine was cultured to give aGSH content of 3.0% compared with the parent content of0.665% (3.5-fold increase; Ikeno et al. 1977), (2) C. utilisresistant to methionine was treated with UV, X-radiation,

and NTG to select a mutant resistant to both sulfite andethionine and the intracellular GSH content increased from0.52% to 4.0% (6.7-fold increase; Kono et al. 1977), (3) S.cerevisiae TRZ-6 with resistance to 1,2,4-triazole and

NaN3 had a GSH content of 2.5% (4.0-fold increase;Hamada et al. 1983). The production of GSH byfermentative methods is summarized in Table 3. Generally,a GSH content of 35% can be obtained using thesemutants. A particularly high level of GSH content, 9.5%,was also reported in S. cerevisiae (Ishii and Miyajima1989).

236



5/10

Table3

Anoverviewofliterature

onthefermentativeproductionofglutath

ionebymicroorganisms.TTemperature,

tculturetime,superscriptrresistant,su

perscriptssensitive

Strain

Phenotype

Carbon

source

NitrogensourceSpecialnutrient

T (C)

t (h)

GSHa

GSH

(mgl1)b

Extraction

Recovery

rate

ScaleReference

C.

tropicalisFERM-P

3368

Wildtype

3.0%Glucose(NH4)2SO4,

(NH4)2HPO4

N

icotinamide

30

24

550

FlaskTannoetal.

(1979)

C.

tropicalisFERM-P

4173

Wildtype

4.2%molas-

ses

Urea,

NH4H2PO4

30

14

0.73

Water(90C)

FlaskSuzukiandMat-

subayashi

(1980)

C.

tropicalisPK233

Wildtype

5%ethanol,

0.5%glucose

(NH4)2SO4

C

asaminoacid,

biotin30

96

60(extracel-

lular)

FlaskNipponZeon

Co.(1983)

C.

tropicalis239D5

Ethioniner

,

sulfiter

3.0%glucose(NH4)2SO4,

(NH4)2HPO4

N

icotinamide

30

24

5

FlaskHamazawaetal.

(1998)

C.

utilisIFO1086

Wildtype

3.0%glucose(NH4)2HPO4

Y

eastextract,corn

s

teepliquor,L-cys-

t

eine

30

20

2.2

250

Water(95C),

Cu2+-H2S

70

20lTannoetal.

(1976)

C.

utilisn74-8

Ethionines

3.0%glucose(NH4)2HPO4

Y

eastextract

30

15

3.0

450

H2SO4,

Cu

2+-

H2S

74.6

70lIkenoetal.

(1977)

C.

utilisFERM-P6907Ethioniner

,

sulfiter

3.0%glucose(NH4)2SO4

30

24

4.5

680

H2SO4,

Cu2O-

H2S

70

200lKohjinCo.

(1984)

C.

utilisFERM-P7396Ethioniner

,

sulfiter


302630

5.0

735

200lHinoetal.

(1985)

C.

utilisWSH02-06

Wildtype


24

28

2.5

385

7l

Weietal.

(2003a)

S.cerevisiaeIAM4207

3%methanol,

2%molasses

(NH4)2SO4

30

48

1.85

80

H2SO4,

Cu2O-

H2S

4050

25lMiyamotoetal.

(1977)

S.cerevisiaeTRZ-6

1,2,4-triazoler,

NaN3

r

6%molasses

(NH4)2SO4,

NH4H2PO4

C

ornsteepliquor

30

24

3.5

860

H2SO4,

Cu2O-

H2S

70

2l

Hamadaetal.

(1983)

S.cerevisiaeR-3

Amider,

indophenolr

6%molasses

(NH4)2SO4,

NH4H2PO4

C

ornsteepliquor

30

24

680

H2SO4,

Cu2O-

H2S

70

2l

Kawamuraetal.

(1985)

S.cerevisiaeK-2

Deficientinga-

lactoseforma-

tion

5%sucrose

Urea,(NH4)

2SO4,

(NH4)

2HPO4

G

alactose

33

24

2,700

FlaskNomuraetal.

(1985)

S.cerevisiae

Glucose

Urea

B

iotin

30

40

9.5

4,320

125lIshiiandMiyaji-

ma(1989)

S.cerevisiaeKY6186

Molasses

(NH4)2SO4,

NH4H2PO4

30

60

3.7

120

m3

SakatoandTa-

naka(1992)

S.cerevisiae

Glucose

(NH4)2SO4

30

7.6

300

3l

Alfafaraetal.

(1993)

S.cystinovolensFERM-

P3831

Wildtype

2%cornsteep

liquor

(NH4)2SO4

Y

eastextract,DL-

m

ethionine,L-cys-

t

eine

30

32

116

H2SO4

(95C)

FlaskMiwaandTaji-

ma(1978b)

237



6/10

There are few reports on the selection of prokaryoticGSH producers. Immobilized methylglyoxal (MG)-resis-tant E. coli produced GSH to levels 1.6-fold higher thanthat of the control in the presence of MG. Presumably thestress induced by the toxic MG increased the production ofGSH, which functioned as a detoxifier (Murata et al.1981c). Selenite-resistant mutants of E. coli producedhigher amounts of GSH (ranging from 9% to 173%,

compared with the wild type; Schmidt and Konetzka1986). Following the observation that GSH was present ina blue-green bacterium (Fahey et al. 1978), someresearchers were interested in finding GSH producersamong green algae or cyanobacteria. These bacteriadisplayed GSH-related different characteristics, comparedwith yeast or E. coli. For instance, the cyanobacterium

Phormidium lapideum produced GSH in the presence ofprecursor amino acids and ATP; and the ATP wasefficiently regenerated from ADP using light as anexternal energy source. GSH was also produced underdark conditions without ATP, when ATP was presumablyregenerated by a respiratory system active in the dark

(Sawa et al. 1986). The production of GSH by the greenalga Dunaliella sp. was also of industrial interest. A GSHcontent of 2.38% was achieved in a medium containingonly inorganic nutrients (Yamaoka and Takimura 1990).Although the relatively low total biomass concentrationlimited the final GSH concentration, this study providedan alternative way to produce GSH, or high-GSH-contentfood additives.

Process optimization

The ultimate goal of the biotechnological production of

GSH is to achieve a high total GSH concentration throughincreasing the intracellular GSH content and cell density.While the intracellular GSH content was improvedsignificantly by mutagenesis, the enhancement of cellconcentration could be achieved by process optimizationand control. The intracellular GSH content might also bemaximized under the control of a suitable cultivationstrategy.

Selection of nutrients in the culture medium is usuallyimportant for the fermentative production of GSH and theconcentration of nutrients should also be optimized.Central composition experimental design has been appliedto examine the effects of certain nutrients (glucose,

peptone, KH2PO4, biotin, cysteine) on the cell growthand intracellular GSH content of S. cerevisiae S-8H. Themean of total GSH concentration obtained at the optimalconditions was 160.1 mg l1, which was nearly 2-fold thatof the control (Udeh and Achremowicz 1997). BoxBehnken design and response surface methodology havealso been used to optimize the concentration of glucose,

peptone, and MgSO4 in the production of GSH by S.cerevisiae CCRC 21727. The derived neural networkmodels predicted cell growth and GSH production more

precisely than the second-order response surface models(Liu et al. 1999).S

train

Phenotype

Carbon

source

NitrogensourceSpecialnutrient

T (C)

t (h)

GSHa

GSH

(mgl1)b

Extraction

Recovery

rate

ScaleReference

Methylomonasmetha-

nolvorensFERM-P

3330

Wildtype

Methanol

(NH4)2SO4

30

48

1.82

50

Water(90C),

chromotog-

raphy

80

25lMiyamotoetal.

(1978)

ProteusmirabilisIFO

3849

Wildtype

1.0%glucosePeptone,

NaNH4HPO4

Serine,H3BO3

31

36

180(extracel-

lular)

Chromotography61

2l

AjinomotoCo.

(1983)

Dunaliellasp.(

irradia-

tion1200lx)

Wildtype

CO2

NaNO3

24

144

2.38

FlaskYamaokaand

Takimura

(1990)

Phormidiumlapideum

(irradiation2500lx)

Wildtype

CO3

2,

HCO3

48

24

0.15

FlaskOchiai(1987)

aTheintracellularGSHcontent,sho

wnasthemasspercentageofGSHinth

etotaldrycellweight(%)

bThetotalGSHconcentrationinthefermentationmedium,whichequalsthe

intracellularGSHcontentmultipliedby

thedrycellconcentration

Table3

(continued)

238 http://www.paper.edu.cn


7/10

Fed-batch culture is one of the most efficient methods toachieve a high cell density of yeast culture. However, thespecific growth rate () during fed-batch culture should becarefully controlled, to avoid a decrease in intracellularGSH content. Based on the mass balance, together withthe relationship between and the specific GSH

production rate, a simple mathematic model was devel-oped, to determine the optimal profile of in a yeast fed-

batch culture (Shimizu et al. 1991). An ideal profile offor maximizing the production of GSH was realized bymanipulating the glucose-feeding rate with the extendedKalman filter and a programmed-controller/feedback-compensator system. As a consequence, the maximum

production of GSH was 41% higher than that of thecontrol (Shimizu et al. 1991). Furthermore, a fuzzy logiccontroller was developed to control the ethanol concen-tration in fed-batch cultures of S. cerevisiae, to maximizeGSH production. Moreover, when of cells in the GSH

production phase was adjusted to maintain a specific GSHproduction rate of 6.2 mg g1 h1, the total GSHconcentration achieved was 56% higher than that of the

control, where was kept at a constant level throughoutthe process (Alfafara et al. 1993). Alternatively, using afeed-forward and a feed-back control system based on theon-line data of oxygen and ethanol concentrations in theexhaust gas, an average of 40% improvement in GSH

production was obtained, compared with the conventionalprogrammed control of exponential fed-batch operationwith S. cerevisiae (Sakato and Tanaka 1992).

Although sugar material was the major substrate in thefermentative production of GSH, the addition of precursoramino acids required for GSH was an easy approach intrials to increase GSH production. L-Cysteine wasconfirmed to be a key amino acid for increasing the

specific GSH production rate, but it showed some growthinhibition in the second growth phase of S. cerevisiaewhen glucose was used as the sole carbon source (Alfafaraet al. 1992a). Therefore, a suitable L-cysteine additionstrategy should be developed to increase GSH productionwithout causing growth inhibition. Alfafara et al. (1992b)found that single-shot addition of L-cysteine was betterthan continuous addition, where the concentration of L-cysteine was maintained at a constant level (Alfafara et al.1992b). Using this knowledge, they developed a mass

balance model-based feeding strategy in which L-cysteinewas added in a single-shot manner to a culture entering theGSH production phase. As a consequence, the specific

GSH production rate increased approximately 2-foldcompared with that of the control (Alfafara et al.1992b). A similar stimulatory effect of L-cysteine onGSH production was observed in recombinant E. coli (Liet al. 1998), where the total GSH concentration and theintracellular GSH content increased by 40% and 100%,respectively, when 9 mM L-cysteine was added to theculture at 12 h. Besides L-cysteine, several other materialswere found to have a stimulatory effect on GSH

production: amino acid supplements (yeast extract, pep-tone) on S. cerevisiae (3.4-fold increase; Watanabe et al.1986), ethanol (26 g l1) on S. cerevisiae (1.4-fold

increase; Kyowa Hakko Kogyo Co. 1984), p-aminoben-zoic acid (200 mg l1) on Hansenula capsuleita (94%increase; Kinoshita et al. 1986), and sodium lactate (10 gl1) on S. cerevisiae (82% increase; Hirakawa et al. 1985).

Genetic/metabolic engineering

Early studies on the properties of GSHI and GSHIIshowed that these enzymes were feedback-inhibited byGSH and GSSG, respectively (Murata and Kimura 1990),indicating that GSHI was the rate-limiting step in GSH

biosynthesis. The genes gshA and gshB encoding GSHI(Murata et al. 1981a) and GSHII (Murata et al. 1983) werecloned and sequenced. Specifically, an E. coli B strainwith GSHI desensitized to feedback inhibition of GSHwas screened and the desensitized GSHI-coding gene

gshA* was cloned (Murata and Kimura 1982). Theintroduction of the recombinant plasmid pGS500 harbor-ing the gshA* and gshB genes into E. coli RC912 resultedin a simultaneous increase in the activities of GSHI (10.0-

fold) and GSHII (14.5-fold; Gushima et al. 1983).Although the intracellular GSH concentration in E. coliRC912 (pGS500) cells only increased 1.3-fold comparedwith the wild type, the intact recombinant E. coli cellswere used as an excellent GSH biosynthesis system, bywhich 5 g l1 GSH was produced in the presence of three

precursor amino acids and ATP. Similar work was done inS. cerevisiae, where the expression of GSHI and GSHIIincreased 1,039-fold and 33-fold, respectively, and theintracellular GSH content increased 2-fold (Ohtake et al.1988, 1989).

The overexpression of gshA and gshB did not lead to asignificant increase in GSH content in either E. coli or S.

cerevisiae. This might be due to: (1) feedback inhibitionon GSHI caused by GSH, or (2) the presence of -GTPdegrading the GSH synthesized intracellularly. Recently,an extremely high intracellular concentration of GSH, upto 140 mM upon addition of 5 mM L-cysteine, wasachieved in Lactococccus lactis NZ9000 expressing the

gshA and gshB from E. coli under the control of a nisin-induced control expression system. This is the highestintracellular GSH content achieved for a bacterium to date(Li et al. 2004). L. lactis is not capable of GSH

biosynthesis and therefore there is no -GTP activity.Furthermore, the feedback inhibition of GSH on GSHIseems not to occur in L. lactis (where the intracellular

GSH concentration of 140 mM achieved was obviouslyhigher than the concentration normally required forfeedback inhibition), although the reason for that remainsunclear. The development of genetic engineering ap-

proaches for GSH production is summarized in Table 4.

Conclusions and perspectives

One topic that is not discussed in this Mini-Review is thesecretion of GSH, a process which might increase the totalGSH concentration in the culture and facilitate the

239



8/10

recovery process. C. tropicalis PK233 (Nippon Zeon Co.1983) and Proteus mirabilis IFO 3849 (Ajinomoto Co.1983) are capable of producing a certain amount of GSHextracellularly. We have shown that the addition of acritical concentration of surfactants enhanced the extra-cellular accumulation of GSH by S. cerevisiae (Wei et al.2003b). We recently showed that C. utilis 02-08 secretedGSH into the medium when the pH of the culture

decreased to pH 1.5 (Nie W et al., unpublished data).Based on this observation, a pH shift strategy wasdeveloped and the total concentration of GSH increased(up to 2.0-fold). If GSH can be produced extracellularly byfood-grade microorganisms, i.e., lactic acid bacteria, theGSH present in the food matrix will no doubt increase thenutritional value, as GSH is considered as a nutraceutical.

Related to another aspect for future studies, previousefforts on GSH production mainly focused on improvingthe activity of the GSH biosynthesis system itself.However, as cysteine availability is usually the limitingstep for GSH biosynthesis, we propose developing afurther metabolic engineering strategy to improve the

cysteine biosynthesis capability, instead of focusing onlyon the GSH biosynthesis pathway. In addition, it is widelyrecognized that GSH plays important roles in oxidativestress resistance. However, this property was not related tothe production of GSH until Kimura et al. (1996) reportedthe cloning and expression of oxidative stress-resistancegenes from a peroxide-resistant S. cerevisiae. Theintroduction of these genes into another S. cerevisiaestrain enhanced GSH production (up to 2.5-fold; Kimuraet al. 1996). This investigation gave a clue how to design anovel metabolic engineering strategy for GSH production.

In conclusion, GSH is successfully produced on anindustrial scale using fermentative methods and serves as

an important pharmaceutical. The range of applicationswhere GSH is employed is expanding in line with thediscovery of new functional roles for this bioactivetripeptide. With the design and/or discovery of novel

producers, or the deregulation of biosynthesis in theknown producers, the biotechnological approach offerssignificant opportunities to further decrease the productioncost of GSH, which will further the application andutilization of GSH.

Acknowledgements The authors thank Dr. Paul W. OToole forcritically reading this manuscript. This study was supported by theNational Science Foundation of China (contract no. 30300009).

References

Ajinomoto Co. (1982a) Production of glutathione. JP patent57,002,698

Ajinomoto Co. (1982b) Production of glutathione. JP patent57,005,699

Ajinomoto Co. (1983) Microbial production of glutathione. JPpatent 58,016,694

Ajinomoto Co. (1984) Production of glutathione. JP patent59,156,298

Table4

Productionofglutathione

byrecombinantmicroorganisms.Formo

reinformationontheproductionofglutathionebyengineeredE.

coliorS.cerev

isiae,refertoMurataand

Kimura(1990)

Hoststrain

Cloningstrategy

GSHinmu

tant

GSHinwildtypeIncrement

(x-fold)

Reference

S.cerevisiae

Astrongpromoter,P8fromS.cerevisiaeYNN27,wasused

toreplacethepromoterof

GSHI,resultingin

plasmidpGRS2518-x

24mgg

1

3mgg

1

7

Tezukaetal.

(1987)

E.

coli

HindIIIdigestedchromosomalDNAofE.

coliwasinsertedintopBR322

andtransformedintoGSH-deficientE.

colitoselectpositiveclones

7.9molg1

(2.4

2mgg

1)

1.3molg

1

(0.5

5mgg

1)

3.4

KimuraandMur-

ata(1983)

E.

coli

DNAfragmentcontainingthestructuralandpromoterregion

ofgshB(encodingGSHII)

wasclonedinsingle,

double,andtriplecopiesintopBR325

Littleimproved

Watanabeetal.

(1986)

E.

colia

ArecombinantbacteriophagecontainingtwocopiesofgshA

andonecopyofgshBofE.

coliwasconstructedtotransfectE.

coli

7.9molm

g1

proteinh

1

Documents

chenjian-15.pdf