Biosynthesis of superoxide dismutase and catalase in chemostat ...

Applied Microbiology Bioteehnology

Appl Microbiol Biotechnol (1987) 26:531--536

© Springer-Verlag 1987

Biosynthesis of superoxide dismutase and catalase in chemostat culture of Saccharomyces cerevisiae Fang-Jen S. Lee and Hosni M. Hassan Departments of Food Science and Microbiology, North Carolina State University, Raleigh, NC 27695, USA

Summary. The effects of oxygen (100%), paraquat (0.5 mM), and copper (0.1 mM) on the growth and the biosynthesis of the antioxidant enzymes, superoxide dismutase (SOD) and catalase, were studied in Saccharomyces cerevisiae grown in glucose-limited chemostat cultures. The effect of dilution rates (D, h - l ) on cell mass, glucose consumption, ethanol production, oxygen uptake, and specific activities of SOD and catalase were also investigated at each steady state. SOD was optimally produced at D-values between 0.22 and 0.26 h-1 in the presence of oxygen or paraquat, and at D-values greater than 0.17 h-~ when copper was used. On the other hand, catalase activity decreased with increasing D-values. However, the presence of copper or 100% oxygen repressed catalase activity at low D-values (D 0.22 h - 1). We also studied the effect of oxygen concentration on the biosynthesis of SOD and catalase at D -- 0.1 h - 1. The data clearly show that synthesis of SOD and catalase, though correlated with changes in oxygen tension, are independent of one another.

Paper Number 10871 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned Offprint requests to: H. M. Hassan

Introduction Oxygen though essential for aerobic growth and survival, is known to be toxic (Fridovich 1975). The toxicity of oxygen is related to its partially reduced intermediates that include the superoxide radical (O 2), hydrogen peroxide (H202), and the hydroxyl radical (OH'). These are extremely reactive compounds which can directly or indirectly cause substantial damage to the delicate structure of living cells (Fridovich 1975; Hassan and Fridovich 1980). Superoxide dismutase (SOD) catalyzes the dismutation of the O ~ radical to generate H202 and 02, while catalase decomposes the H202 generated. The coordinate action of these enzymes keeps the concentrations of intracellular O 2 and H202 very low, and minimizes the risk of their interaction to generate the more toxic OH- (Hassan and Fridovich 1980; McCord and Day 1978). The application of superoxide dismutase as an antioxidant (Michelson and Monod 1975; Michelson 1977), and as an anti-inflammatory and radioprotective drug in medicine (McCord and English 1981; Misra and Fridovich 1976; Petkau 1978) has prompted its commercial production from bovine blood and from genetically engineered microorganisms (Hallewell et al. 1985). In a previous study we have shown that oxygen, copper and paraquat can induce the copperzinc superoxide dismutase (CuZnSOD) in Saccharomyces cerevisiae (Lee and Hassan 1985, 1986). In this study, a chemostat culture was used to determine the optimum conditions for the biosynthesis of CuZnSOD in S. cerevisiae. Data are presented to show the effects of dilution rate, oxygen tension, copper and paraquat on biomass, oxygen uptake, glucose metabolism and on the biosynthe-

532

F.-J. S. Lee and H. M. Hassan: Biosynthesis of superoxide dismutase and catalase

sis of superoxide dismutase and catalase, using a glucose-limited chemostat culture.

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Materials and methods

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Saccharomyces cerevisiae ATCC 4111 was obtained from the American Type Culture Collection.

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Media and chemicals The basal synthetic medium used contained (per liter): (NH4)2SO4, 2.0 g; MgSO4.7 H20, 0.25 g; CaC12.2 H20, 0.05 g; FeSOa.7H20, 3 l-tg; KI, 1 ~tg; ZnSO4.7H20, 4.2 I~g; MnSO4. H20, 0.3 ~tg; CuSO4.5 H20, 0.1 Ilg; biotin, 6.25 I.tg; minositol, 1.25rag; niacin, 0.2511g; p-aminobenzoic acid, 0.125 mg; pyridoxin hydrochloride, 0.25 mg; riboflavin, 0.25 mg; thiamine hydrochloride, 0.25 mg. The basal medium was supplemented per liter with 1.44g glucose, 1.75g Na2HPO4-2 H20 and 13.5 g KH2PO4 to give the basal glucosephosphat (BGP) medium. Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride) was obtained from Sigma.

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Culture conditions The inoculum for each experiment was prepared by transferring a loopful from a slant culture of S. cerevisiae to 50 ml BGP medium and growing the culture overnight at 30°C and 160 rpm. Chemostat culture experiments were performed in a water-jacketed glass culture vessel (5 cm diameter x 35 cm height; total volume - 850 ml) with 400 ml working volume (V. ml). Temperature was controlled at 30°C using a circulating water bath (Fig. 1). The pH of culture at steady state remained at 5.3+0.2 without the use of a pH controller. The culture was mix.ed with a Teflon-coated magnetic stirrer at 300 rpm and the gas flow rate was set at 280 ml/min. The flow of incoming medium was controlled by a peristaltic pump (Buchler Instruments). Flow rate (F, ml/h) was determined by measuring the culture effluent collected during a measured time interval. Steady state for chemostat culture at a given dilution rate ( D = F / V , h - i ) was assumed when the cell density (OD600) remained unchanged within +2% after three changes of the working volume. Samples were removed aseptically through a sampling port at the top of the chemostat vessel. Once the steady state of the culture was established, about 60 ml of the culture was removed using a sterile syringe.

Cell dry weight Cell dry weight was determined from a calibration curve relating OD600 to dry weight, where one OD600=0.266 mg dry cell/ml (Lee and Hassan 1986).

Preparation of cell-free extracts and analytical assays Cells were harvested by centrifugation, and dialysed cell-free extracts were prepared as previously described (Lee and Has-

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Fig. l. Diagram of the chemostat culture used san 1985). Superoxide dismutase was assayed according to McCord and Fridovich (1969) and modified by adding 10 -5 M C N - to inhibit cytochrome oxidase. Catalase was assayed as described by Beers and Sizer (1952). Protein was determined by the method of Lowry et al. (1951) using bovine serum albumin as a standard. Oxygen uptake by whole cells was measured at 30°C using Clark Polarographic Electrode as described (Lee and Hassan 1986).

Determination of glucose, ethanol and acetic acid Glucose was measured using a glucose kit based on glucose oxidase/peroxidase (Sigma Chemical Co.) and ethanol was determined by an ethyl alcohol kit based on alcohol dehydrogenase (Sigma Chemical Co.). Acetic acid was determined by HPLC using a Bio-Rad Aminex HPX-87H column and RI detector (Waters Associates).

Results

Effects of dilution rates and oxygen concentrations on growth, S O D and catalase S. cerevisiae was exposed to air and 100% oxygen during growth in a glucose-limited chemostat culture operated at dilution rates between 0.04 h and 0.4 h-~ (Fig. 2). At each dilution rate, the culture was allowed to reach a steady state before samples were removed. The data show that expo-

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Fig. 2. Effects of dilution rate and oxygen concentration on growth and enzyme biosynthesis in chemostat culture of S. cerevisiae. Cells were grown, in the presence of air ( , ) or in the presence of 100% (11), in the glucose-limited (8 mM) chemostat culture as described in Materials and methods. The cells in the steady state were removed at different cilution rates and immediately assayed for biomass and oxygen uptake. The rate for oxygen uptake is expressed as m m o l / g , h - 1. Dialyzed cell-free extracts were prepared and assayed for SOD and catalase as described in Materials and methods. The supernatants were assayed for glucose and ethanol. A : Biomass, B : Oxygen uptake, C : Glucose, D : Ethanol, E : SOD activity, F : Catalase activity

sure to 100% 02 resulted in a 25--40% decline in cell mass as compared to cells exposed to air (Fig. 2A). The rates of oxygen uptake (mmoles/gram dry wt. cells per hour) for cells grown in the presence of 100% oxygen were, in general, 50--60% less than the rates of oxygen uptake seen with airgrown cells (Fig. 2B). A positive correlation was observed between D. h - 1 and the rate of oxygen uptake for both types of cells. The rates of glucose consumption by air- and 100% O2-grown cells were the same for D-values between 0.05 and 0.3 h - ~. However, at D > 0.3 h - 1, cells grown in 100% 02 consumed glucose at a slightly slower rate than air-grown cells (Fig. 2C). Ethanol accumulation was higher in 100% O2-grown cells than in air-grown cells (Fig. 2D), which may suggest that air-grown cells are more capable of oxidizing the ethanol and produce more cells (Fig. 2A).

The specific activity of SOD was 80 to 160% higher in cells grown in 100% 0 2 than in airgrown cells (Fig. 2E). In the presence of 100% O2, SOD was optimally produced by cells grown at dilution rates between 0.22 and 0.26 h-1 (Fig. 2E). The specific activity of catalase was dramatically repressed in cells exposed to 100% 02 at all Dvalues examined. However, in the presence of air there was an inverse relationship between the specific activity of catalase and the specific dilution rates, D. h - 1 (Fig. 2F).

Effect of transitions in oxygen concentration on 9rowth, SOD and catalase In this part of the study the dilution rate was kept constant at 0 . 1 + 0 . 0 0 5 h -I and transitions in 02

534

F.-J. S. Lee and H, M. Hassan: Biosynthesis of superoxide dismutase and catalase

concentrations were achieved by changing the sparging gases (i.e. nitrogen + air + 100% oxygen --, nitrogen). The culture was allowed to reach steady state after each transition and before samples were taken for analysis (Fig. 3). It is clear that changes in the oxygen concentration in the sparging gases had a significant effect on cell density and the rate of oxygen uptake (Fig. 3A). Growth in the presence of air (20% 02) gave optimal cell biomass and maximum rate of oxygen uptake. Growth in the presence of 100% oxygen resulted in a 35% decrease in cell yield as compared to air-grown cells. As expected, growth in the presence of nitrogen resulted in the lowest cell

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Fig. 3. Effect of changes in oxygen concentration on growth and enzyme biosynthesis in chemostat culture of S. cerevisiae. The yeast were grown in a chemostat culture at D = 0.1___0.005 h - ] , and N2, air, or 100% 02 were supplied at defined periods as shown. The cells were removed at the different steady states and biomass and oxygen uptake (retool/ g . h - t ) were measured. Dialysed cell-free extracts were prepared and assayed for SOD and catalase as described in Materials and methods. The supernatants were assayed for glucose, ethanol and acetic acid. A: Biomass and oxygen uptake; B: Glucose, Ethanol and acetic acid; C: SOD and catalase

Effects of copper and paraquat on growth, SOD and catalase Previous studies (Lee and Hassan 1985) have shown that copper and paraquat can affect cell growth and antioxidant enzyme biosynthesis in batch culture. The addition of 0.1 m M CuSO4 to S. cerevisiae grown in air in chemostat culture at different dilution rates had no significant effect on cellular dry-weight (Fig. 4A vs. Fig. 2A). However, it stimulated SOD biosynthesis at dilution rate >0.17 h -1 and inhibited oxygen uptake and catalase biosynthesis at all dilution rates tested. On the other hand, addition of 0.5 mM paraquat inhibited cellular metabolism, stimulated SOD biosynthesis, and had no significant effect on catalase biosynthesis. Addition of 0.1 mM CuSO4 plus 0.5 mM paraquat inhibited cell mass, oxygen uptake and ethanol utilization, but had a stimulatory effect on SOD biosynthesis greater than that obtained in the presence of CuSO+ or paraquat alone (Fig. 5 vs. Fig. 4).

Discussion

Previous studies on SOD and catalase biosynthesis in S. cerevisiae were performed in batch cultures (Lee and Hassan 1985, 1986). Interpretation

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Fig, 4. Effects of copper or paraquat on growth and enzyme biosynthesis in chemostat culture of S. cerevisiae. Cells were grown in the presence of 0.1 m M copper ( 0 ) or in the presence of 0.5 m M paraquat (ram) in the glucose-limited chemostat culture as described in Materials and methods. Sample preparation and assays were the same as in Fig. 2. A : Biornass, B : Oxygen uptake, C : Glucose, D: Ethanol, E: SOD activity, F: Catalase activity

of the results is not straightforward because of the complex physiological changes that take place in non-steady state populations (Kjeldgaard et al. 1958). In the present study, these difficulties were overcome by using steady-state chemostat cultures of S. cerevisiae. This system allowed us to study the effects of oxidative stress under balanced growth conditions over a wide range of specific growth rates in a constant environment and for an extended period of time. S. cerevisiae, when grown in a glucose-limited chemostat culture, revealed systematic variations in response to changes in dilution rates and in oxygen concentrations. At D < 0 . 0 5 h - I the cell yield decreased due to the fact that a high proportion of the energy available to the cells was used for maintenance instead of growth. Catalase activity was highest at D = 0 . 0 5 h -~. At D-values > 0.05 h - ~ but < 0.2 h - 1 glucose was completely oxidized and only trace amounts of ethanol were found. Ethanol accumulated in cultures grown at

D_> 0.22 h -1, where also the cell yield declined, suggesting incomplete oxidation of the glucose utilized probably due to some unknown nutrient limitation. A positive correlation was observed between the dilution rate and oxygen uptake. A strong positive correlation was also found between the rate of oxygen uptake and SOD. These results are similar to those reported in E. coli (Hassan and Fridovich 1977a). Growth in the presence of 100% oxygen was inhibitory to all growth parameters examined except for SOD activity which showed at least a two-fold increase relative to air-grown cells. It is clear that 100% oxygen must have caused damage to essential cell functions and that SOD was induced in an attempt to protect the cells against oxygen toxicity (Fridovich 1975). The data also demonstrate that the cells were capable of adjusting to sudden changes in their oxygen content (Fig. 3) and that SOD plays an important role as evidenced by the cell's ability to modulate SOD

F.-J. S. Lee and H. M. Hassan: Biosynthesis of superoxide dismutase and catalase

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Acknowledgements. This work was supported in-part by DMP8609239 from the National Science Foundation and 86-G00719 from N. C. Biotechnology Center.

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Fig. 5. Effect of copper plus paraquat on growth and enzyme biosynthesis in chemostat culture of S. cerevisiae. Cells were grown supplemented with 0.1 mM copper plus 0.5 mM paraquat in the glucose-limited chemostat culture and were assayed as in Fig. 2. A : Biomass and oxygen uptake; B : Glucose and ethanol; C: SOD and catalase

activity to meet the oxidative stress imposed on them. Paraquat is known to generate superoxide radicals in E. coli (Hassan and Fridovich 1977b) and in S. cerevisiae (Lee and Hassan 1985). It has also been shown that copper induces SOD in S. cerevisiae (Lee and Hassan 1985, Greco et al. 1986). Our present findings, using chemostat culture, support and extend this thesis by showing that the effect of paraquat and copper on SOD biosynthesis is a function of increasing dilution rate (i.e. specific growth rate of the cells). Addition of copper and paraquat to the medium inhibited cellular metabolism, but increased the level of SOD to a greater extent than when each was added separately. The effect was neither synergistic nor additive at dilution rate > 0.2 h-1, which supports our previous conclusion derived from batch culture studies (Lee and Hassan 1985). Our present findings clearly indicate that it is possible to increase the level of SOD in S. cerevisiae by optimizing the specific growth rate, oxygen tension and by adding copper and paraquat to cells grown in a chemostat culture.

Beers RF, Sizer IW (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195:133--140 Fridovich I (1975) Superoxide dismutase. Annu Rev. Biochem 44:147-- 195 Greco MA, Magner W, Hrab D, Kosman D (1986) Superoxide dismutase and the physiological response to copper in S. cerevisiae. In: Rotilio G (ed) Superoxide and superoxide dismutase in chemistry, biology and medicine. Elsevier Sci Pub Co Inc, pp 296--299 Hallewell RA, Masiarz FR, Najarian RC, Puma JP, Quiraga MR, Randolph A, Sanchez-Peescador R, Sacndella C J, Smith B, Steimer KS, Mullenbach GT (1985) Human Cu/ Zn superoxide dismutase cDNA: isolation of synthesizing high levels of active or inactive enzyme from an expression library. Nucleic Acids Res 6:2017--2023 Hassan HM, Fridovich I (1977a) Physiological function of superoxide dismutase in glucose-limited chemostat cultures of Escherichia coil J Bacteriol 130:805--811 Hassan HM, Fridovich I (1977b) Regulation of the synthesis of superoxide dismutase in Escherichia coli: induction by methyl viologen. J Biol Chem 252:7667--7672 Hassan HM, Fridovich I (1980) Superoxide dismutase: Detoxication of a Free Radical. In: Jakoby WB (ed) Enzymatic Basis ofDetoxication. Vol 1. Academic Press Inc, pp 311 -- 332 Kjelgaard NO, Maaloe O, Schaechter M (1958) The transition between different physiological states during balanced growth of Salmonella typhimurium. J Gen Microbiol 19:607--617 Lee FJ, Hassan HM (1985) Biosynthesis of superoxide dismutase in Saccharomyces cerevisiae: Effects of paraquat and copper. J Free Radicals Biol Med 1:319--325 Lee FJ, Hassan HM (1986) Biosynthesis of superoxide dismutase and catalase in Saccharomyces cerevisiae: Effects of oxygen and cytochrome c deficiency. J Industrial Microbiol 1:187--193 Lowry OH, Rosebrough N J, Farr AL, Randall RJ (1951) Protein measurement with the folin phenol reagent, J Biol Chem 193:265--275 McCord JM, Fridovich I (1969) Superoxide dismutase: an enzymatic function of erythrocuperein. J Biol Chem 244:6049--6055 McCord JM, Day Jr ED (1978) Superoxide dependent production of hydroxyl radical catalysed by iron-EDTA complex. FEBS Lett 86:139--140 McCord JM, English D (1981) Superoxide dismutase: An antiinflammatory drug. In: Holcember JS (ed) Enzyme as drugs. John Wiley & Sons, New York, pp 353--365 Michelson AM, Monod J (1975) Superoxide dismutase and it's application as an oxidation inhibitor. US Patent 3,920,521 Michelson AM (1977) Superoxide dismutase and it's application as an oxidation inhibitor. US Patent 4,029,819 Misra HP, Fridovich I (1976) Superoxide dismutase and the oxygen enhancement of radiation lethality. Arch of Biochem and Biophys 176:577-581 Petkau A (1978) Radiation protection by superoxide dismutase. Photochem and Photobiol 28:765--774 Received February 3, 1987/Revised April 7, 1987