Oxidative stress response of a recombinant Aspergillus niger to exogenous menadione and H2O2 addition

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Abstract

A recombinant strain of Aspergillus niger (B1-D), engineered to produce the marker protein hen egg white lysozyme (HEWL), was investigated with regard to its susceptibility to oxidative stress. The culture response to oxidative stress, produced either by menadione (MD) or by H2O2, was characterised in terms of the intracellular activities of two key defensive enzymes, catalase (CAT) and superoxide dismutase (SOD), in the presence or absence of a range of vitamin supplements.

Addition of either ascorbate (Vit C) or α-tocopherol (Vit E) resulted in different responses of A. niger cultures to exogenous H2O2 (1 mM). In the presence of Vit C the activity of SOD was increased by 50% compared to control cultures, whereas incorporation of Vit E appeared to have a “protective” effect against reactive oxygen species (ROS).

Continuous addition of menadione (1.2 mmole l−1 h−1) to submerged cultures of A. niger in the bioreactor resulted in an increase of CAT and SOD activities during the exponential phase of growth, whereas enzyme levels remained almost constant during that phase in control cultures. Comparison of these results with the response to oxidative stress caused by addition of exogenous H2O2 and elevated dissolved oxygen tension (DOT) [1] shows that these three stressors are quite distinct.

Introduction

All aerobically growing micro-organisms encounter toxic, reactive oxygen species (ROS) including the superoxide radical (O2.−), H2O2 and the hydroxyl radical (OH.). The production of oxygen free radicals is now accepted to be an inevitable consequence of aerobic metabolism: ROS are mainly produced by “leakage” of electrons from the cellular electron-transport chain onto molecular oxygen, and they have been implicated in damage to virtually all types of major biologic macromolecules [2], [3], [4]. Consequently, aerobic cells have evolved strategies for defence against damage induced by such species, including defensive enzymes, such as superoxide dismutases, catalases and peroxidases, as well as by non-enzymic antioxidants, such as ascorbate and tocopherols [2], [5]. Under normal physiological conditions, the toxic effects of ROS are minimised by these enzymic and non-enzymic antioxidants. However, under stressful conditions, oxidant levels may increase to overwhelm the antioxidants, resulting in cell damage. So far, the oxidative stress response which protects organisms from the deleterious effects of ROS has been extensively examined in bacteria, including E. coli, and S. typhimurium [6], [7] as well as in yeast [8], [9], [10]. In contrast, despite their continued industrial significance surprisingly few data are available on the antioxidative defence systems of filamentous fungi [1], [11], [12], [13], [14]. However, inadequate mixing and mass transfer, due to the viscous character of fungal bioprocesses [15], may expose fungal cells to variations in DOT, including transient exposure to regions of high DOT, which in turn may lead to oxidative stress effects.

Oxidative stress in cells is usually studied by exposing the cells, mostly in shake-flask cultures, to high concentrations of pro-oxidants for a short period of time by a single addition of the stressing chemical, and comparing the enzyme activities obtained in stressed cultures and control cultures a few hours after the addition of the stressing compound. Oxidants such as H2O2 or superoxide radical generating agents are frequently used in this regard [5], [6], [8], [9], [11]. Clearly, this method of studying cellular defensive response to oxidative stress has theoretical and practical weaknesses, in particular, it represents a “snapshot” of the response, which provides no information at all on the dynamics of the defensive process, secondly, it takes no account of fluctuations in the levels of such enzymes over the life cycle (or growth phases) of the micro-organisms. A key practical issue is just how well the single point addition of an exogenous chemical agent actually simulates the impact of an oxidative stress such as exposure to the elevated DOT, which fungal cells might encounter in industrial submerged culture process as a consequence of the poor mixing referred to above.

By contrast, the present study on oxidative stress induced by continuous exogenous addition of MD uses cultures grown under closely controlled conditions in bioreactors, in combination with exposure to oxidative stress over sufficient periods of time to allow possible effects on growth rate and enzyme activities to become manifest. Also, enzyme levels were assessed not only at time of the addition of the stressor and a certain time thereafter but during the whole course of the culture growth. With this approach we were able to get a more comprehensive picture of how fungal cultures cope with oxidative stress in terms of activities of the defensive enzymes CAT and SOD. In particular, the variation with culture growth phase in the activity of these key enzymes can be examined, and an assessment can be made of the relative impact of the various oxidative “stressors” (MD, H2O2 and elevated DOT).

Medium composition is of major importance in fermentation industry. Microorganisms are often more resistant toward stress if grown in complex media [16]. One explanation for this is that certain media components facilitate coping with stress for the microbial cells. In animal cell cultures the levels of extracellular non-enzymic antioxidants are important [17]. Their provision is achieved normally by serum supplementation. The importance of these serum components is crucial, as deprivation of serum causes an increase of intracellular superoxide generation and lipid peroxidation [17]. In general, microorganisms are assumed to be less sensitive than animal cell cultures, however, there are also indications that vitamin supplementation affects their response to oxidative stress [18], [19]. Antioxidant vitamins have been known to act as powerful free radical quenchers in several biologic systems through their ability to accept an additional electron and become radicals themselves. These vitamin radicals are more stable, and can be regenerated, thus, avoiding possible cell damage [2].

In this study on oxidative stress in A. niger we investigated the effects of medium supplementation with vitamins (ascorbic acid and α-tocopherol) on the capability of the strain to cope with oxidative stress induced by addition of exogenous H2O2 and menadione. We examined these effects in terms of the activities of CAT and SOD in shake flask cultures with carefully controlled inocula in a defined physiological condition, derived from the bioreactor.

Section snippets

Micro-organism and culture conditions

A. niger strain B1-D (kindly supplied by Prof. D. Archer, IFR Norwich, U.K.) containing the hen egg white lysozyme (HEWL) cDNA gene fused to a glucoamylase (glaA) promoter was used [20]. The organism was stored as a freeze-dried spore suspension at 4°C. Spore suspensions were produced as described earlier [1]. A defined medium was used with glucose (30 g l−1) as a carbon-source and energy source. The composition of the medium was as described by Wongwicharn et al. [21]. PPG 2025 (BDH, Poole,

Effect of antioxidant supplementation on the oxidative stress response of A. niger shake flask cultures to menadione and H2O2

Mid exponential A. niger cultures, grown in the bioreactor, transferred to shake-flasks, and exposed to a single addition of MD neither showed a significant increase in the activities of CAT and SOD nor was the growth inhibited. The CDW, obtained 21 h after the addition of MD, was similar in cultures exposed to MD (11.19 ± 0.50, 11.40 ± 0.34, and 11.79 ± 0.40 g liter−1, for 50, 500, and 1000 μM, respectively) or H2O2 (11.17 ± 0.50 g liter−1, for 1 mM) and untreated control cultures (11.33 ±

Conclusions

A. niger batch cultures responded to chemical oxidant stressors, H2O2 or MD, immediately after starting continuous addition, whereas gassing with oxygen enriched air did not significantly effect CAT and SOD activities in the exponential phase. Contrasting to the continuous addition of H2O2, which increased only CAT activities in this culture phase, the continuous addition of MD elevated additionally, after a short lag phase, SOD activity. Maximum activities of CAT and SOD were obtained in the

Acknowledgements

M. Kreiner thanks the Austrian Science Foundation (FWF) for an Erwin-Schrödinger-Auslandsstipendium as a postdoctoral fellow at the University of Strathclyde. We also thank Prof. David Archer (I.F.R., Norwich, U. K.) for the provision of the strain used in this study, along with Mr. Walter McEwan for technical assistance.

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