Appl Microbiol Biotechnol (2004) 66: 233–242 DOI 10.1007/s00253-004-1751-y

MINI-REVIEW

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 historic developments and technological achievements in the biotechnological production of glutathione in the past 30 years. Glutathione is the most abundant non-protein thiol compound present in living organisms. It is used as a pharmaceutical compound and can be used in food additives and the cosmetic industries. Glutathione can be produced using enzymatic methods in the presence of ATP and its three precursor amino acids (L-glutamic acid, Lcysteine, glycine). Alternatively, glutathione can be produced by direct fermentative methods using sugar as a starting material. In the latter method, Saccharomyces cerevisiae and Candida utilis are currently used to produce glutathione on an industrial scale. At the molecular level, the genes gshA and gshB, which encode the enzymes γglutamylcysteine synthetase and glutathione synthetase, respectively, have been cloned from Escherichia coli and over-expressed in E. coli, S. cerevisiae, and Lactococcus lactis. It is anticipated that, with the design and/or discovery of novel producers, the biotechnological production of glutathione will be further improved to expand the application range of this physiologically and medically important tripeptide.

Introduction Glutathione (γ-glutamyl-L-cysteinylglycine, GSH) is the most abundant non-protein thiol compound widely Y. Li (*) . G. Wei . J. Chen The Key Laboratory of Industrial Biotechnology, Ministry of Education; School of Biotechnology, Southern Yangtze University, 170 Huihe Road, Wuxi, 214036, People’s Republic of China e-mail: [email protected] Tel.: +86-510-5885727 Fax: +86-510-5888301 G. Wei School of Life Science, Soochow University, Suzhou, 215007, People’s Republic of China

distributed in living organisms and, predominantly, in eukaryotic cells (Meister and Anderson 1983). While over 90% of the glutathione is normally present in the reduced form GSH, several additional forms of glutathione are present in (microbial) cells, tissues, and plasmas. Glutathione disulfide GSSG (oxidized glutathione), formed upon 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, GSH may occur in other forms of mixed disulfides, for example, GS-S-CoA, GS-S-Cys (Penninckx 2002), and GS-S-protein which is formed via glutathionylation. Although GSH has been found to be involved in many physiological processes and to play various important roles, the major and general functions of GSH can be summarized into three major ways, i.e. serving as antioxidant, immunity booster, and detoxifier in higher eukaryotic organisms (Pastore et al. 2003). First, the strong electron-donating capability of GSH and the relatively high intracellular concentration (up to millimolar levels) enable the maintenance of a reducing cellular environment. This makes GSH an important antioxidant for protecting DNA, proteins, and other biomolecules against oxidative damage generated by, for example, reactive oxygen species. Second, GSH plays an important role in immune function via white blood cell production and is one of the most potent anti-viral agents known. Finally, GSH can be conjugated to exogenous electrophiles and diverse xenobiotics by glutathione-S-transferase to accomplish detoxification. GSH is thus considered to be one of the most powerful, versatile, and important self-generated defense molecules. In humans, GSH deficiency has been linked to a number of disease states: HIV infection, liver cirrhosis, pulmonary diseases, gastrointestinal and pancreatic inflammations, diabetes, neurodegenerative diseases, and aging (Wu et al. 2004). GSH is widely used as a pharmaceutical compound and has the potential to be used in food additives and in the cosmetic industries (Sies 1999), given that the price can be further decreased by improving production methods.

234

While the physiological roles of GSH in human and animal tissues, in plant cells, and in microbial cells have been extensively explored and described (Carmel-Harel and Storz 2000; Penninckx and Elskens 1993; Penninckx 2000, 2002), reviews covering the biotechnological production of this medically important tripeptide are scarce. One reason might be that much of the outcome of research on the production of GSH is patented, due to the great economic interest. The latest reviews describing the production of GSH were published nearly 10 years ago (Murata 1989, 1994; Murata and Kimura 1990), with special emphasis on the production of GSH by genetically engineered Escherichia coli or Saccharomyces cerevisiae. In this Mini-Review, we aim to provide a summary of the historic developments and technological achievements in the biotechnological production of GSH in the past 30 years. Patents relevant to enzymatic and fermentative production of GSH are cited, to show the diversity of glutathione-producing approaches. Furthermore, future perspectives on the biotechnological production of glutathione are given.

Production of glutathione: a brief historic overview GSH was discovered in ethanol-extract of baker’s yeast in 1888 and was referred to as “philothion.” It was subsequently renamed glutathione follow the establishment of its molecular structure in 1921 (Penninckx and Elskens 1993). With the discovery of the presence of GSH in many living organisms, solvent extraction of GSH from animal or plant tissues was exploited as a preparative approach. However, the limited raw materials available and the relatively low intracellular content of GSH made the end-product expensive, thus hampering its practical application. Nearly 70 years ago, it was demonstrated that glutathione could be synthesized by a chemical method (Harington and Mead 1935). The introduction and removal of sulfhydryl protective agents on a cysteine residue were crucial steps in the chemical synthesis of GSH (Douglas 1989). Although this process was comTable 1 Development of the biotechnological production of GSH

Fig. 1 Overview of GSH-related metabolism pathways and the general physiological roles played by GSH in microorganisms. Cys cysteine, Gly glycine, Glu glutamate, G6PDH glucose-6-phosphate dehydrogenase, GPx glutathione peroxidase, GR glutathione reductase, GRX glutaredoxin, GSHI γ-glutamylcysteine synthetase, GSHII glutathione synthetase, GTP γ-glutamyltransferase, ROOH peroxides, ROH reduced peroxides, S–S disulfide bond

mercialized in the 1950s, chemically synthesized GSH was an optically inactive (racemic) mixture of the D- and Lisomers. As only the L-form is physiologically active, an optical resolution is required to separate the L-form from its D-isomer. Following the observation of the biosynthesis of GSH in an isolated liver and the characterization of the biosynthetic pathway for GSH (Bloch 1949), explorations on the enzymatic and fermentative production of GSH were pursued. GSH is synthesized in two consecutive ATPdependent reactions (Meister and Anderson 1983). The dipeptide γ-glutamylcysteine (γ-GC) is first synthesized from L-glutamic acid and L-cysteine by γ-glutamylcysteine synthetase (also known as glutamate-cysteine ligase, EC 6.3.2.2, GSHI). In the second step, catalyzed by glutathione synthetase (EC 6.3.2.3, GSHII), glycine is added to the C-terminal site of γ-GC to form GSH. Generally, the activity of GSHI is feedback-inhibited by GSH (but not GSSG) to avoid over-accumulation of GSH, which is of physiological significance (Richman and Meister 1975). Meanwhile, cellular GSH can be degraded by γ-

Research interest in GSH production

Period

Breeding of yeast (predominantly S. cerevisiae and Candida utilis) for glutathione 1976–1985, production, using either fermentative or enzymatic methods 1998–present Purification of glutathione from biological systems 1976–1987, 1993–1998 Selection of bacteria to produce glutathione using enzymatic methods 1978–1991 Construction of ATP regeneration systems 1978–1982 Molecular biology of GSH biosynthesis in E. coli and construction of recombinant 1982–1990 E. coli to produce GSH Construction of recombinant S. cerevisiae to produce GSH 1986–1990, 1996–1998 Optimization and control of glutathione production process 1991–1994, 1997–present Production of glutathione by lactic acid bacteria 2001–present

2,517

7.5 –

30

ATP Not necessary Glycolysis (glucose)

Achromobacter lacticum FERM-P 7401 Corynebacterium glutamicum ATCC 21171 (frozen cells treated with surfactant, xylene)

Dried yeast

Proteus mirabilis (cell-free extract) P. mirabilis IFO 3849 P. vulgaris (crude GSHI, GSHII enzymes modified by Nethylmaleimide Phormidium lapideum (2,500 lx)

Light used as an external energy source

Acetate kinase prepared from E. coli B ATCC 23226

ATP

Glycolysis (glucose) Dextran-N-[(6-aminohexyl)carbamoylmethyl] ATP ATP, glucose ATP ATP

E. coli ATCC 11303

E. coli B ATCC 23226 (crude enzymes of GSHI, GSHII)

ATP

E. coli (treated with surfactant, xylene)

Brevibacterium ammoniagenes ATCC 21170 (frozen cells treated with surfactant, xylene)

325

7.3 5

35

47

37 37 37

37

37

37

37 37

7.5 12

8.5 2 9.15 12 9.15 12

1.5 mg g−1 wet cell

1,320 280 480

46.3

2,700

7.4 – 7.0 3

2,714

560 2,456

7.4 6

6.0 16 7.4 6

9,060

11.1

7.3 Continuous 8,989

7.5 18

350 240

37

30

6.0 16 7.5 5

GSH (mg l−1)

7.3 15

S. cerevisiae IFO 0304 (treated by sodium lauryl sulfate)

37 30

T pH t (°C) (h)

37

ATP supply

S. cerevisiae IFO 0021 (treated by sodium dodecyl sulfate and β-1,3-glucanase) S. glyoxalphilus CDA-5 (treated by sodium dodecyl sulfate, immobilized) S. cerevisiae 500 (immobilized crude enzymes of GSHI, Immobilized carbamylGSHII) phosphokinase from Streptococcus faecalis R 600 S. cerevisiae (immobilized GSHI, GSHII)

ATP regeneration

Not necessary Glycolysis (glucose) Glycolysis (glucose) Glycolysis (glucose) Glycolysis (glucose) ATP

C. krusei IFO 0011 S. cerevisiae IFO 2044 (immobilized)

GSH biosynthesis

61.4 1.3 –

3.0

35.2

58.9

2.2 49.0

82

58.6

97.6

98.4

24.1

1.4 7.8

Yield on Cys (%)

Sawa et al. (1986)

Ajinomoto Co. (1984) Ajinomoto Co. (1982a) Ajinomoto Co. (1982b)

Chibata et al. (1979a)

Kyowa Hakko Kogyo Co. (1985a) Fujio et al. (1985)

Miyamoto and Miwa (1977) Yokozeki et al. (1985) Kyowa Hakko Kogyo Co. (1985b)

Miwa and Tajima (1978a) Miwa (1976)

Miwa (1978)

Takesue et al. (1979)

Yokozeki et al. (1985) Chibata et al. (1979b)

Reference

Table 2 An overview of the literature on the enzymatic production of glutathione by microorganisms. Enzymatic production of glutathione uses a reaction mixture containing L-glutamic acid, L-cysteine, and glycine

235

236

glutamyltranspeptidase (γ-GTP) to transfer the γ-glutamyl moiety (Meister and Anderson 1983), which is important in amino acid transport. To overproduce GSH using either enzyme/intact cell biocatalysis or fermentation, the release of GSHI from feedback inhibition by GSH and/or the inactivation or deficiency of γ-GTP are necessary (Murata 1994). Figure 1 gives a biochemical overview, showing the biosynthetic and metabolic pathways and illustrating the general physiological roles of GSH. Table 1 summarizes the development of the biotechnological production of GSH. Research on fermentative and enzymatic production of GSH was very active between 1976 and 1985 in Japan; and fermentative production of GSH by yeast was commercialized in the early 1980s. Since then, the number of patents on the production of GSH declined dramatically. To date, the enzymatic production of GSH has not been commercialized because of the relatively high production cost.

only feasible approach to date. The reaction catalyzed by acetate kinase in E. coli cells was explored as an ATP generation system to be coupled with GSH biosynthesis reactions, resulting in the successful production of GSH (Langer et al. 1976). However, the acetyl phosphate substrate required for this approach was unstable and expensive. Murata and co-workers considered the glycolytic pathway of S. cerevisiae to be the simplest and the most capable system for regenerating sufficient ATP for GSH biosynthesis (Murata et al. 1981b). When the economic supply of ATP was no longer a problem, the low activities of GSHI and GSHII became the limiting factors in GSH biosynthesis. The requirement to enhance the activities of GSHI and GSHII accelerated the application of molecular cloning and genetic engineering approaches in GSH biosynthesis.

Fermentative production of glutathione Enzymatic production of glutathione

GSH producer screening

One of the well studied approaches to glutathione production is the enzymatic method. In principle, the essential elements to constitute an enzymatic synthesis system include: GSHI, GSHII, precursor amino acids (Lglutamic acid, L-cysteine, glycine), ATP, necessary cofactors (Mg2+) to maintain the activities of GSHI and GSHII, and a suitable pH (usually pH 7.5). Table 2 gives an overview of the enzymatic production of GSH using either intact cells or crude enzymes. It can be generally concluded from Table 2 that the addition of ATP is not necessary 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 of glycolysis compensates the consumption of ATP. Also, increasing the cell wall permeability (induced by freezing– thawing and/or treatment with surfactants or enzymes) enhances enzymatic production significantly when intact cells are used. The requirement for ATP in the enzymatic production of glutathione makes this process difficult to scale-up, since it is impractical from an economic point of view to add ATP directly on an industrial scale. Accordingly, it would be of industrial interest to construct a highly efficient ATP regeneration system, which can be briefly defined as a system in which ATP-requiring reactions are coupled with ATP-producing reactions. ATP regeneration systems can be categorized into self-coupling systems which work within one organism and co-coupling systems which are constructed between two or more organisms. When a cocoupling ATP regeneration system is used, the efficiency of ATP/ADP transport across the respective cell membranes should be carefully taken into account. Self-coupling ATP regeneration systems have not been intensively investigated, since it is difficult to simultaneously improve the activities of both GSH biosynthesis and ATP regeneration in one organism (Shimosaka et al. 1982). The co-coupling ATP regeneration system is the

The advantage of the enzymatic production of GSH is that a high concentration (up to 9 g l−1; Table 2) can be achieved. However, since the usage of three precursor amino acids increases the production cost, the fermentative production of glutathione using sugar materials as substrates has been extensively studied and is the major commercial method currently used. S. cerevisiae and Candida utilis are the most commonly used microorganisms on an industrial scale; and the GSH contents of the wild-type strains are usually high (0.1–1.0% dry cell weight). Therefore, these two microorganisms were chosen as targets for mutagenesis. The physical or chemical mutagenesis methods used included UV, Xradiation, γ-radiation, and N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment, while resistance to compounds such as glutathione analogues (methionine, ethionine), 1,2,4-triazole, sodium cyanide, and sulfite was used to screen GSH over-producers. The mechanism for the selection, in most cases, was to disrupt or release the feedback inhibition of GSH on GSHI. Several examples illustrate how powerful the screening strategy was: (1) C. utilis n74-8 sensitive to DL-ethionine was cultured to give a GSH content of 3.0% compared with the parent content of 0.665% (3.5-fold increase; Ikeno et al. 1977), (2) C. utilis resistant to methionine was treated with UV, X-radiation, and NTG to select a mutant resistant to both sulfite and ethionine and the intracellular GSH content increased from 0.52% 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 by fermentative methods is summarized in Table 3. Generally, a GSH content of 3–5% can be obtained using these mutants. A particularly high level of GSH content, 9.5%, was also reported in S. cerevisiae (Ishii and Miyajima 1989).

Wild type

C. tropicalis FERM-P 3368 C. tropicalis FERM-P 4173

Glucose

2% corn steep (NH4)2SO4 liquor

S. cerevisiae

S. cystinovolens FERM- Wild type P 3831

(NH4)2SO4, NH4H2PO4 (NH4)2SO4

Molasses

S. cerevisiae KY6186

S. cerevisiae

S. cerevisiae K-2

S. cerevisiae R-3

S. cerevisiae TRZ-6

3% methanol, (NH4)2SO4 2% molasses 1,2,4-triazoler, 6% molasses (NH4)2SO4, NaN3r NH4H2PO4 Amider, 6% molasses (NH4)2SO4, indophenolr NH4H2PO4 Deficient in ga- 5% sucrose Urea, (NH4) lactose forma2SO4, (NH4) tion 2HPO4 Glucose Urea Corn steep liquor

Yeast extract, DLmethionine, L-cysteine

30

30

30

30

33

Galactose

Biotin

30

Corn steep liquor

30

30

24

2.8% glucose (NH4)2SO4

32

7.6

60 3.7

40 9.5

24

24

24 3.5

48 1.85

28 2.5

30→26 30 5.0

24 4.5

15 3.0

20 2.2

24 5

3.0% glucose (NH4)2SO4

S. cerevisiae IAM 4207

C. utilis FERM-P 6907 Ethioniner, sulfiter C. utilis FERM-P 7396 Ethioniner, sulfiter C. utilis WSH 02-06 Wild type

30

30

30

96

14 0.73

24

116

300

4,320

2,700

680

860

80

385

735

680

450

250

60 (extra cellular)

550

t GSHa GSH (h) (mg l−1)b

30

Ethionines

C. utilis n74-8

Yeast extract, corn steep liquor, L-cysteine Yeast extract

Nicotinamide

Casamino acid, biotin 30

30

30

T (°C)

3.0% glucose (NH4)2SO4

3.0% glucose (NH4)2HPO4

Ethioniner, sulfiter Wild type

C. tropicalis 239D5

C. utilis IFO1086

5% ethanol, (NH4)2SO4 0.5% glucose 3.0% glucose (NH4)2SO4, (NH4)2HPO4 3.0% glucose (NH4)2HPO4

Wild type

Nicotinamide

Nitrogen source Special nutrient

3.0% Glucose (NH4)2SO4, (NH4)2HPO4 4.2% molas- Urea, ses NH4H2PO4

Carbon source

C. tropicalis PK233

Wild type

Phenotype

Strain

H2SO4 (95°C)

H2SO4, Cu2OH2S H2SO4, Cu2OH2S H2SO4, Cu2OH2S

H2SO4, Cu2+H2S H2SO4, Cu2OH2S

Water (95°C), Cu2+-H2S

Water (90°C)

Extraction

70

70

40–50

70

74.6

70

125 l Ishii and Miyajima (1989) 120 Sakato and Tam3 naka (1992) 3 l Alfafara et al. (1993) Flask Miwa and Tajima (1978b)

70 l Ikeno et al. (1977) 200 l Kohjin Co. (1984) 200 l Hino et al. (1985) 7 l Wei et al. (2003a) 25 l Miyamoto et al. (1977) 2 l Hamada et al. (1983) 2 l Kawamura et al. (1985) Flask Nomura et al. (1985)

Flask Tanno et al. (1979) Flask Suzuki and Matsubayashi (1980) Flask Nippon Zeon Co. (1983) Flask Hamazawa et al. (1998) 20 l Tanno et al. (1976)

Recovery Scale Reference rate

Table 3 An overview of literature on the fermentative production of glutathione by microorganisms. T Temperature, t culture time, superscript r resistant, superscript s sensitive

237

Ajinomoto Co. (1983) Flask Yamaoka and Takimura (1990) Flask Ochiai (1987)

b

144

24 0.15

24

48

Wild type

CO32−, HCO3− Wild type Phormidium lapideum (irradiation 2500 lx)

a

36 – 31 Serine, H3BO3

1.0% glucose Peptone, NaNH4HPO4 NaNO3 CO2 Wild type

(NH4)2SO4 Methanol Wild type

The intracellular GSH content, shown as the mass percentage of GSH in the total dry cell weight (%) The total GSH concentration in the fermentation medium, which equals the intracellular GSH content multiplied by the dry cell concentration

2l

25 l Miyamoto et al. (1978)

Water (90°C), 80 chromotography 180 (extracel- Chromotography 61 lular) 2.38 48 1.82 30

50

t GSHa GSH (h) (mg l−1)b

Methylomonas methanolvorens FERM-P 3330 Proteus mirabilis IFO 3849 Dunaliella sp.(irradiation 1200 lx)

Table 3 (continued)

Carbon source Phenotype Strain

Nitrogen source Special nutrient

T (°C)

Extraction

Recovery Scale Reference rate

238

There are few reports on the selection of prokaryotic GSH producers. Immobilized methylglyoxal (MG)-resistant E. coli produced GSH to levels 1.6-fold higher than that of the control in the presence of MG. Presumably the stress induced by the toxic MG increased the production of GSH, which functioned as a detoxifier (Murata et al. 1981c). Selenite-resistant mutants of E. coli produced higher amounts of GSH (ranging from 9% to 173%, compared with the wild type; Schmidt and Konetzka 1986). Following the observation that GSH was present in a blue-green bacterium (Fahey et al. 1978), some researchers were interested in finding GSH producers among green algae or cyanobacteria. These bacteria displayed GSH-related different characteristics, compared with yeast or E. coli. For instance, the cyanobacterium Phormidium lapideum produced GSH in the presence of precursor amino acids and ATP; and the ATP was efficiently regenerated from ADP using light as an external energy source. GSH was also produced under dark conditions without ATP, when ATP was presumably regenerated by a respiratory system active in the dark (Sawa et al. 1986). The production of GSH by the green alga Dunaliella sp. was also of industrial interest. A GSH content of 2.38% was achieved in a medium containing only inorganic nutrients (Yamaoka and Takimura 1990). Although the relatively low total biomass concentration limited the final GSH concentration, this study provided an alternative way to produce GSH, or high-GSH-content food additives. Process optimization The ultimate goal of the biotechnological production of GSH is to achieve a high total GSH concentration through increasing the intracellular GSH content and cell density. While the intracellular GSH content was improved significantly by mutagenesis, the enhancement of cell concentration could be achieved by process optimization and control. The intracellular GSH content might also be maximized under the control of a suitable cultivation strategy. Selection of nutrients in the culture medium is usually important for the fermentative production of GSH and the concentration of nutrients should also be optimized. Central composition experimental design has been applied to examine the effects of certain nutrients (glucose, peptone, KH2PO4, biotin, cysteine) on the cell growth and intracellular GSH content of S. cerevisiae S-8H. The mean of total GSH concentration obtained at the optimal conditions was 160.1 mg l−1, which was nearly 2-fold that of the control (Udeh and Achremowicz 1997). Box– Behnken design and response surface methodology have also been used to optimize the concentration of glucose, peptone, and MgSO4 in the production of GSH by S. cerevisiae CCRC 21727. The derived neural network models predicted cell growth and GSH production more precisely than the second-order response surface models (Liu et al. 1999).

239

Fed-batch culture is one of the most efficient methods to achieve a high cell density of yeast culture. However, the specific growth rate (μ) during fed-batch culture should be carefully controlled, to avoid a decrease in intracellular GSH content. Based on the mass balance, together with the relationship between μ and the specific GSH production rate, a simple mathematic model was developed, to determine the optimal profile of μ in a yeast fedbatch culture (Shimizu et al. 1991). An ideal profile of μ for maximizing the production of GSH was realized by manipulating the glucose-feeding rate with the extended Kalman filter and a programmed-controller/feedbackcompensator system. As a consequence, the maximum production of GSH was 41% higher than that of the control (Shimizu et al. 1991). Furthermore, a fuzzy logic controller was developed to control the ethanol concentration in fed-batch cultures of S. cerevisiae, to maximize GSH production. Moreover, when μ of cells in the GSH production phase was adjusted to maintain a specific GSH production rate of 6.2 mg g−1 h−1, the total GSH concentration achieved was 56% higher than that of the control, where μ was kept at a constant level throughout the process (Alfafara et al. 1993). Alternatively, using a feed-forward and a feed-back control system based on the on-line data of oxygen and ethanol concentrations in the exhaust gas, an average of 40% improvement in GSH production was obtained, compared with the conventional programmed control of exponential fed-batch operation with S. cerevisiae (Sakato and Tanaka 1992). Although sugar material was the major substrate in the fermentative production of GSH, the addition of precursor amino acids required for GSH was an easy approach in trials to increase GSH production. L-Cysteine was confirmed to be a key amino acid for increasing the specific GSH production rate, but it showed some growth inhibition in the second growth phase of S. cerevisiae when glucose was used as the sole carbon source (Alfafara et al. 1992a). Therefore, a suitable L-cysteine addition strategy should be developed to increase GSH production without causing growth inhibition. Alfafara et al. (1992b) found that single-shot addition of L-cysteine was better than continuous addition, where the concentration of Lcysteine 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-cysteine was added in a single-shot manner to a culture entering the GSH production phase. As a consequence, the specific GSH production rate increased approximately 2-fold compared with that of the control (Alfafara et al. 1992b). A similar stimulatory effect of L-cysteine on GSH production was observed in recombinant E. coli (Li et al. 1998), where the total GSH concentration and the intracellular GSH content increased by 40% and 100%, respectively, when 9 mM L-cysteine was added to the culture at 12 h. Besides L-cysteine, several other materials were found to have a stimulatory effect on GSH production: amino acid supplements (yeast extract, peptone) on S. cerevisiae (3.4-fold increase; Watanabe et al. 1986), ethanol (26 g l−1) on S. cerevisiae (1.4-fold

increase; Kyowa Hakko Kogyo Co. 1984), p-aminobenzoic acid (200 mg l−1) on Hansenula capsuleita (94% increase; Kinoshita et al. 1986), and sodium lactate (10 g l−1) on S. cerevisiae (82% increase; Hirakawa et al. 1985).

Genetic/metabolic engineering Early studies on the properties of GSHI and GSHII showed that these enzymes were feedback-inhibited by GSH 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) were cloned and sequenced. Specifically, an E. coli B strain with GSHI desensitized to feedback inhibition of GSH was screened and the desensitized GSHI-coding gene gshA* was cloned (Murata and Kimura 1982). The introduction of the recombinant plasmid pGS500 harboring the gshA* and gshB genes into E. coli RC912 resulted in a simultaneous increase in the activities of GSHI (10.0fold) and GSHII (14.5-fold; Gushima et al. 1983). Although the intracellular GSH concentration in E. coli RC912 (pGS500) cells only increased 1.3-fold compared with the wild type, the intact recombinant E. coli cells were used as an excellent GSH biosynthesis system, by which 5 g l−1 GSH was produced in the presence of three precursor amino acids and ATP. Similar work was done in S. cerevisiae, where the expression of GSHI and GSHII increased 1,039-fold and 33-fold, respectively, and the intracellular GSH content increased 2-fold (Ohtake et al. 1988, 1989). The overexpression of gshA and gshB did not lead to a significant increase in GSH content in either E. coli or S. cerevisiae. This might be due to: (1) feedback inhibition on GSHI caused by GSH, or (2) the presence of γ-GTP degrading the GSH synthesized intracellularly. Recently, an extremely high intracellular concentration of GSH, up to 140 mM upon addition of 5 mM L-cysteine, was achieved in Lactococccus lactis NZ9000 expressing the gshA and gshB from E. coli under the control of a nisininduced control expression system. This is the highest intracellular 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 γ-GTP activity. Furthermore, the feedback inhibition of GSH on GSHI seems not to occur in L. lactis (where the intracellular GSH concentration of 140 mM achieved was obviously higher than the concentration normally required for feedback inhibition), although the reason for that remains unclear. The development of genetic engineering approaches for GSH production is summarized in Table 4.

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

a

L. lactis

S. cerevisiae

S. cerevisiae

E. colia

E. coli

E. coli

Production of glutathione was performed in a reaction mixture containing precursor amino acids, ATP, and acetyl phosphate

– 358 nmol mg−1 Cannot produce protein (55 mg g−1)

– –

–

<0.1 μmol mg−1 78 protein h−1 – 2.5 7.9 μmol mg−1 protein h−1 –

3.4

1.3 μmol g−1 (0.55 mg g−1) – 7.9 μmol g−1 (2.42 mg g−1) –

Little improved

Tezuka et al. (1987) Kimura and Murata (1983) Watanabe et al. (1986) Matsuyama et al. (1989) Kimura et al. (1996) Omura et al. (1998) Li et al. (2004) A strong promoter, P8 from S. cerevisiae YNN27, was used to replace the promoter of GSHI, resulting in plasmid pGRS2518-x HindIII digested chromosomal DNA of E. coli was inserted into pBR322 and transformed into GSH-deficient E. coli to select positive clones DNA fragment containing the structural and promoter region of gshB (encoding GSHII) was cloned in single, double, and triple copies into pBR325 A recombinant bacteriophage containing two copies of gshA and one copy of gshB of E. coli was constructed to transfect E. coli Oxidative stress-resistance genes from a peroxide-resistant S. cerevisiae were cloned and transformed into S. cerevisiae SHY2 Single point mutations in the transcriptional activator of MET gene, [Pro215]Met4, [Ser156] Met4, resulted in an enhanced transcription of MET genes gshA and gshB genes, encoding GSHI and GSHII, respectively, were amplified from E. coli TG1 chromosomal DNA and inserted into pNZ8148 to generate pNZ3203 S. cerevisiae

7 3 mg g−1 24 mg g−1

Reference GSH in wild type Increment (x-fold) GSH in mutant Cloning strategy Host strain

Table 4 Production of glutathione by recombinant microorganisms. For more information on the production of glutathione by engineered E. coli or S. cerevisiae, refer to Murata and Kimura (1990)

240

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 GSH extracellularly. We have shown that the addition of a critical concentration of surfactants enhanced the extracellular accumulation of GSH by S. cerevisiae (Wei et al. 2003b). We recently showed that C. utilis 02-08 secreted GSH 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 was developed and the total concentration of GSH increased (up to 2.0-fold). If GSH can be produced extracellularly by food-grade microorganisms, i.e., lactic acid bacteria, the GSH present in the food matrix will no doubt increase the nutritional value, as GSH is considered as a nutraceutical. Related to another aspect for future studies, previous efforts on GSH production mainly focused on improving the activity of the GSH biosynthesis system itself. However, as cysteine availability is usually the limiting step for GSH biosynthesis, we propose developing a further metabolic engineering strategy to improve the cysteine biosynthesis capability, instead of focusing only on the GSH biosynthesis pathway. In addition, it is widely recognized that GSH plays important roles in oxidative stress resistance. However, this property was not related to the production of GSH until Kimura et al. (1996) reported the cloning and expression of oxidative stress-resistance genes from a peroxide-resistant S. cerevisiae. The introduction of these genes into another S. cerevisiae strain enhanced GSH production (up to 2.5-fold; Kimura et al. 1996). This investigation gave a clue how to design a novel metabolic engineering strategy for GSH production. In conclusion, GSH is successfully produced on an industrial scale using fermentative methods and serves as an important pharmaceutical. The range of applications where GSH is employed is expanding in line with the discovery of new functional roles for this bioactive tripeptide. With the design and/or discovery of novel producers, or the deregulation of biosynthesis in the known producers, the biotechnological approach offers significant opportunities to further decrease the production cost of GSH, which will further the application and utilization of GSH. Acknowledgements The authors thank Dr. Paul W. O’Toole for critically reading this manuscript. This study was supported by the National Science Foundation of China (contract no. 30300009).

References Ajinomoto Co. (1982a) Production of glutathione. JP patent 57,002,698 Ajinomoto Co. (1982b) Production of glutathione. JP patent 57,005,699 Ajinomoto Co. (1983) Microbial production of glutathione. JP patent 58,016,694 Ajinomoto Co. (1984) Production of glutathione. JP patent 59,156,298

241 Alfafara CG, Kanda A, Shioi T, Shimizu H, Shioya S, Suga K (1992a) Effect of amino acids on glutathione production by Saccharomyces cerevisiae. Appl Microbiol Biotechnol 36:538– 540 Alfafara CG, Miura K, Shimizu H, Shioya S, Suga K (1992b) Cysteine addition strategy for maximum glutathione production in fed-batch culture of Saccharomyces cerevisiae. Appl Microbiol Biotechnol 37:141–146 Alfafara CG, Miura K, Shimizu H, Shioya S, Suga K, Suzuki K (1993) Fuzzy control of ethanol concentration and its application to maximum glutathione production in yeast fed batch culture. Biotechnol Bioeng 41:493–501 Bloch K (1949) The synthesis of glutathione in isolated liver. J Biol Chem 179:1245–1254 Carmel-Harel O, Storz G (2000) Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu Rev Microbiol 54:439–461 Chibata I, Kato J, Murata K (1979a) Glutathione. JP patent 54,122,793 Chibata I, Kato J, Murata K (1979b) Glutathione. JP patent 54,138,190 Douglas KT (1989) Chemical synthesis of glutathione and analogs. Coenzymes Cofactors 3:243–279 Fahey RC, Brown WC, Adams WB, Worsham MB (1978) Occurrence of glutathione in bacteria. J Bacteriol 133:1126– 1129 Fujio T, Hayashi M, Tomiyoshi Y, Ozaki A (1985) Compound from its precursor using the enzymic activity of a bacterium. EP patent 146,265 Gushima H, Miya T, Murata K, Kimura A (1983) Construction of glutathione-producing strains of Escherichia coli B by recombinant DNA techniques. J Appl Biochem 5:43–52 Hamada S, Tanaka H, Sakato K (1983) Glutathione. EP patent 79,241 Hamazawa K, Maegawa H, Furue S (1998) NAD and glutathione high content yeast. JP patent 10,191,963 Harington CR, Mead TH (1935) Synthesis of glutathione. Biochem J 29:1602–1611 Hino T, Harada M, Maekawa H (1985) Production of highglutathione-containing yeast cells. JP patent 60,156,379 Hirakawa K, Nomura K, Kato M (1985) Lactic acid for high yield glutathione production by yeasts. JP patent 60,244,284 Ikeno Y, Tanno K, Omori I, Yamada R (1977) Glutathione. JP patent 52,087,296 Ishii S, Miyajima R (1989) Glutathione manufacture by cultivation of Saccharomyces in a synthetic medium. JP patent 1,141,591 Kawamura M, Hamada S, Sakado K (1985) Glutathione by fermentation. JP patent 60,248,199 Kimura A, Murata K (1983) Microorganisms of the genus Escherichia, hybrid DNA for use in their production and the use of the microorganisms in the preparation of glutathione. EP patent 71,486 Kimura H, Inoe Y, Kobayashi S (1996) Glutathione manufacture with recombinant Saccharomyces. JP patent 8,070,884 Kinoshita K, Machida M, Oka S, Yamamoto Y, Tomikanehara H (1986) Manufacture of yeast cells containing high glutathione. JP patent 61,192,282 Kohjin Co. (1984) Glutathione by fermentation. JP patent 59,151,894 Kono G, Harada M, Sugisaki K, Nishida M (1977) High glutathione-containing yeast. JP patent 52,125,687 Kyowa Hakko Kogyo Co. (1984) Yield increase in glutathione produced by yeasts. JP patent 59,034,899 Kyowa Hakko Kogyo Co. (1985a) Microbial production of glutathione. JP patent 60,027,396 Kyowa Hakko Kogyo Co. (1985b) Production of glutathione. JP patent 60,027,397 Langer RS, Hamilton BK, Gardner CR (1976) Enzymatic regeneration of ATP. AIChE J 22:1079–1090

Li Y, Chen J, Mao YY, Lun SY, Koo YM (1998) Effect of additives and fed-batch culture strategies on the production of glutathione by recombinant Escherichia coli. Process Biochem 33:709–714 Li Y, Hugenholtz J, Sybesma W, Abee T, Molenaar D (2004) Using Lactococcus lactis for glutathione overproduction. Appl Microbiol Biotechnol (in press) Liu CH, Hwang C, Liao CC (1999) Medium optimization for glutathione production by Saccharomyces cerevisiae. Process Biochem 34:17–23 Matsuyama A, Nakano E, Watabe K, Murata K, Kimura H (1989) Recombinant DNA encoding gamma-glutamylcysteine synthetase and glutathione synthetase, its preparation, and use for manufacturing glutathione with Escherichia. JP patent 1,228,473 Meister A, Anderson ME (1983) Glutathione. Annu Rev Biochem 52:711–760 Miwa N (1976) Glutathione. JP patent 51,144,789 Miwa N (1978) Production of glutathione by fermentation. JP patent 53,094,090 Miwa N, Tajima S (1978a) Production of glutathione by fermentation. JP patent 53,094,088 Miwa N, Tajima S (1978b) Production of glutathione by fermentation. JP patent 53,094,089 Miyamoto I, Miwa N (1977) Production of glutathione by immobilized glutathione synthetase. JP patent 52,051,089 Miyamoto S, Mitsuba N, Takemoto H (1977) Glutathione by cultivating yeast on methanol. JP patent 52,156,994 Miyamoto T, Miwa N, Takemoto T (1978) Glutathione. JP patent 53,009,393 Murata K (1989) Immobilization, chemical and industrial application, and biosynthetic preparation of glutathione and related compounds. Coenzymes Cofactors 3:187–242 Murata K (1994) Glutathione and its derivatives: produced by recombinant Escherichia coli and Saccharomyces cerevisiae. Bioprocess Technol 19:159–183 Murata K, Kimura A (1982) Some properties of glutathione biosynthesis-deficient mutants of Escherichia coli B. J Gen Microbiol 128:1047–1052 Murata K, Kimura A (1990) Overproduction of glutathione and its derivatives by genetically engineered microbial cells. Biotechnol Adv 8:59–96 Murata K, Tani K, Kato J, Chibata I (1981a) Glutathione production by immobilized Saccharomyces cerevisiae cells containing an ATP regeneration system. Eur J Appl Microbiol Biotechnol 11:72–77 Murata K, Tani K, Kato J, Chibata I (1981b) Glycolytic pathway as an ATP generation system and its application to the production of glutathione and NADP. Enzyme Microb Technol 3:233–242 Murata K, Tani K, Kato J, Chibata I (1981c) Isolation of Escherichia coli B mutant deficient in glutathione biosynthesis. Agric Biol Chem 45:2131–2132 Murata K, Miya T, Gushima H, Kimura A (1983) Cloning and amplification of a gene for glutathione synthetase in Escherichia coli B. Agric Biol Chem 47:1381–1383 Nippon Zeon Co. (1983) Glutathione production. JP patent 58,146,294 Nomura K, Sakaguchi S, Hirakawa K, Kato M (1985) Glutathione by fermentation. JP patent 60,244,299 Ochiai H (1987) Production of glutathione by photofermentation: use of thermophilic cyanobacteria. Bio Ind 4:189–197 Ohtake Y, Watanabe K, Tezuka H, Ogata T, Yabuuchi S, Murata K, Kimura A (1988) The expression of gamma-glutamylcysteine synthetase gene of Escherichia coli in Saccharomyces cerevisiae. Agric Biol Chem 52:2753–2762 Ohtake Y, Watanabe K, Tezuka H, Ogata T, Yabuuchi S, Murata K, Kimura A (1989) Expression of glutathione synthetase gene of Escherichia coli B in Saccharomyces cerevisiae. J Ferment Bioeng 68:390–399 Omura F, Maemura H, Shibano y (1998) Recombinant preparation of glutathione using Saccharomyces cerevisiae mutants having single point mutations in gene MET4 that impair the transcriptional repression of MET genes. JP patent 10,033,161

242 Pastore A, Federici G, Bertini E, Piemonte F (2003) Analysis of glutathione: implication in redox and detoxification. Clin Chim Acta 333:19–39 Penninckx M (2000) A short review on the role of glutathione in the response of yeasts to nutritional, environmental, and oxidative stresses. Enzyme Microb Technol 26:737–742 Penninckx MJ (2002) An overview on glutathione in Saccharomyces versus non-conventional yeasts. FEMS Yeast Res 2:295– 305 Penninckx MJ, Elskens MT (1993) Metabolism and functions of glutathione in micro-organisms. Adv Microb Physiol 34:239– 301 Richman PG, Meister A (1975) Regulation of gamma-glutamycystein synthetase b nonallosteric feedback inhibition by glutathione. J Biol Chem 250:1422 Sakato K, Tanaka H (1992) Advanced control of glutathione fermentation process. Biotechnol Bioeng 40:904–912 Sawa Y, Shindo H, Nishimura S, Ochiai H (1986) Photosynthetic glutathione production using intact cyanobacterial cells. Agric Biol Chem 50:1361–1363 Schmidt MG, Konetzka WA (1986) Glutathione overproduction by selenite resistant Escherichia coli. Can J Microbiol 32:825–827 Shimizu H, Araki K, Shioya S, Suga K (1991) Optimal production of glutathione by controlling the specific growth rate of yeast in fed-batch culture. Biotechnol Bioeng 38:196–205 Shimosaka M, Fukuda Y, Murata K, Kimura A (1982) Application of hybrid plasmids carrying glycolysis genes to ATP production by Escherichia coli. J Bacteriol 152:98–103 Sies H (1999) Glutathione and its role in cellular functions. Free Radic Biol Med 27:916–921 Suzuki Y, Matsubayashi T (1980) Glutathione. JP patent 55,000,019

Takesue H, Fujii K, Miura Y (1979) Glutathione. JP patent 54,086,691 Tanno K, Omori I, Yamada R, Ikeno Y (1976) Glutathione. JP patent 51,139,685 Tanno K, Omori I, Yamada R (1979) Nicotinamide adenine dinucleotide and glutathione. JP patent 54,107,594 Tezuka H, Otake Y, Yabuchi S, Kimura H (1987) A novel Saccharomyces promoter and its use in glutathione biosynthesis. JP patent 62,275,685 Udeh KO, Achremowicz B (1997) High-glutathione containing yeast: optimization of production. Acta Microbiol Pol 46:105– 114 Watanabe K, Yamano Y, Murata K, Kimura A (1986) Glutathione production by Escherichia coli cells with hybrid plasmid containing tandemly polymerized genes for glutathione synthetase. Appl Microbiol Biotechnol 24:375–378 Watanabe K, Kato N, Yoshida N (1989) Glutathione, its manufacture with yeast, and the effects of complex amino acids in medium. JP patent 1,148,181 Wei G, Li Y, Du G, Chen J (2003a) Application of a two-stage temperature control strategy for enhanced glutathione production in the batch fermentation by Candida utilis. Biotechnol Lett 25:887–890 Wei G, Li Y, Du G, Chen J (2003b) Effect of surfactants on extracellular accumulation of glutathione by Saccharomyces cerevisiae. Process Biochem 38:1133–1138 Wu G, Fang Y-Z, Yang S, Lupton JR, Turner ND (2004) Glutathione metabolism and its implications for health. J Nutr 134:489–492 Yamaoka Y, Takimura O (1990) Manufacture of glutathione with green algae Dunaliella. JP patent 2,234,691 Yokozeki K, Takeuchi H, Hirose Y (1985) Glutathione. JP patent 60,160,894