The roles of the alternative NADH dehydrogenases during oxidative stress in cultures of the filamentous fungus Aspergillus niger

Despite the importance of filamentous fungi in the biotechnology industry, little is known about their metabolism under the stressful conditions experienced in typical production fermenters. In the present study, oxygen enrichment was used to recreate an industrial batch process, and the effects of the increasing dissolved oxygen tension were studied as regards the cellular metabolism. It was found that elevated dissolved oxygen tension led to an oxidatively stressful environment, as detailed by rapid initial increases in reactive oxygen species (ROS) concentrations and antioxidant enzyme activities. Intracellular protein concentrations also decreased in oxygenated cultures; this appeared to be concomitant with a decrease in the adenosine-5'-triphosphate (ATP) pool in these cultures. Oxygenated cultures showed early senescence and death compared to aerated control cultures. Despite earlier studies proposing various mechanisms for such findings in fungal cultures subjected to oxidative stress, these findings can best be explained by the fact that in such cultures the activity of alternative NADH dehydrogenases was significantly increased, which served to maintain lower ROS concentrations throughout the duration of the process but in doing so also reduced the ability of the organism to create a proton motive force by which to drive ATP synthesis. The findings of the present study help further our understanding of the central roles of these highly conserved enzymes within fungal metabolism under oxidative stress.     

Elevated temperature effects on the oxidant/antioxidant balance in submerged batch cultures of the filamentous fungus Aspergillus niger B1-D

In the present study the relationship between oxidative stress and elevated culture temperature was examined in an industrially relevant fungal culture, Aspergillus niger B1-D. For the first time, both the intracellular levels of the main stressor species (superoxide radical [O(2) (.-)]) and activities of cellular defensive enzymes (superoxide dismutase [SOD], catalase [CAT], and glutathione peroxide [GPx]) were quantified at varying temperature (25, 30, 35, 40 degrees C) to more fully characterize culture response in different growth phases. Elevated culture temperature led to increased O(2) (.-) levels in various culture phases. In the exponential phase this was due to an enhanced generation of O(2) (.-), whereas in stationary phase a decreased dismutation rate may also have contributed. CAT activities generally increased with culture temperature, whereas GPx activity changed little as temperature rose, indicating that GPx played only a minor role in destroying H(2)O(2) in this A. niger. The combination of elevated temperature (35 degrees C) and increased O(2) supply (50% enrichment) led to decreased levels of O(2) (.-) compared to the cultivation at 35 degrees C gassed with air, probably due to enhanced activity of the alternative fungal respiratory pathway. Our findings indicate that while elevated cultivation temperature does clearly induce oxidative stress events, mechanistically, it does so by a rather more complex route than previous studies indicate. Elevated temperature caused a marked disparity in the activities of SOD and CAT, very distinct from the integrated increase in activity of these enzymes in response to oxidative stress.     

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger.

Intracellular pH homeostasis in the filamentous fungus Aspergillus niger was measured in real time by 31P NMR during perfusion in the NMR tube of fungal biomass immobilized in Ca2+-alginate beads. The fungus maintained constant cytoplasmic pH (pH(cyt)) and vacuolar pH (pH(vac)) values of 7.6 and 6.2, respectively, when the extracellular pH (pH(ex)) was varied between 1.5 and 7.0 in the presence of citrate. Intracellular metabolism did not collapse until a Delta pH over the cytoplasmic membrane of 6.6-6.7 was reached (pH(ex) 0.7-0.8). Maintenance of these large pH differences was possible without increased respiration compared to pH(ex) 5.8. Perfusion in the presence of various hexoses and pentoses (pH(ex) 5.8) revealed that the magnitude of Delta pH values over the cytoplasmic and vacuolar membrane could be linked to the carbon catabolite repressing properties of the carbon source. Also, larger Delta pH values coincided with a higher degree of respiration and increased accumulation of polyphosphate. Addition of protonophore (carbonyl cyanide m-chlorophenylhydrazone, CCCP) to the perfusion buffer led to decreased ATP levels, increased respiration and a partial (1 microm CCCP), transient (2 microm CCCP) or permanent (10 microm CCCP) collapse of the vacuolar membrane Delta pH. Nonlethal levels of the metabolic inhibitor azide (N3-, 0.1 mm) caused a transient decrease in pH(cyt) that was closely paralleled by a transient vacuolar acidification. Vacuolar H+ influx in response to cytoplasmic acidification, also observed during extreme medium acidification, indicates a role in pH homeostasis for this organelle. Finally, 31P NMR spectra of citric acid producing A. niger mycelium showed that despite a combination of low pH(ex) (1.8) and a high acid-secreting capacity, pH(cyt) and pH(vac) values were still well maintained (pH 7.5 and 6.4, respectively).     

Physiological responses of chemostat cultures of Aspergillus niger (B1-D) to simulated and actual oxidative stress

The physiological responses of chemostat cultures of the filamentous fungus, Aspergillus niger (B1-D) to simulated and actual oxidative stress, imposed respectively by addition of exogenous menadione (MD; a superoxide radical generating reagent) and gassing the culture with oxygen enriched air (25%, 50%, 75%, and 100% [v/v]), were examined. Changes in the levels of intracellular superoxide anions and defensive enzyme activities, such as catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), were monitored, together with glutathione and respiratory activity in both the dynamic phase and when a new steady state was established. Culture response to MD addition was distinct from that upon exposure to enriched oxygen conditions, in that MD caused elevated levels of intracellular protein, whereas oxygen enrichment caused reduced protein content, especially at low dilution rates. An unexpectedly low level of superoxide radical was found in oxygen-enriched steady-state cultures (>/=50%) at a range of dilution rates, which was not caused by elevated SOD activity. Under these conditions, it was noted that the ratio of rotenone-insensitive/total respiration increased, suggesting increased activity of the alternative respiratory pathway. This may have had the effect of reducing the endogenous generation of superoxide radicals under oxygen rich conditions, but also may have reduced the ATP yield due to the non-proton-pumping nature of the alternative respiratory pathway. Thus, the negative culture effects noted in many studies at high oxygen levels may not simply be due to elevated endogenous superoxide generation, but could be in part due to the consequences of metabolic changes in the culture that seek to minimize superoxide generation. The dynamic culture response was characterized by rapid elevation of intracellular superoxide anions and associated protective enzymes, especially SOD, and was clearly distinct from the adaptive response just described.     

The thioredoxin system of the filamentous fungus Aspergillus nidulans: impact on development and oxidative stress response

Redox regulation has been shown to be of increasing importance for many cellular processes. Here, redox homeostasis was addressed in Aspergillus nidulans, an important model organism for fundamental biological questions such as development, gene regulation or the regulation of the production of secondary metabolites. We describe the characterization of a thioredoxin system from the filamentous fungus A. nidulans. The A. nidulans thioredoxin A (AnTrxA) is an 11.6-kDa protein with a characteristic thioredoxin active site motif (WCGPC) encoded by the trxA gene. The corresponding thioredoxin reductase (AnTrxR), encoded by the trxR gene, represents a homodimeric flavoprotein with a native molecular mass of 72.2 kDa. When combined in vitro, the in Escherichia coli overproduced recombinant proteins AnTrxA and AnTrxR were able to reduce insulin and oxidized glutathione in an NADPH-dependent manner indicating that this in vitro redox system is functional. Moreover, we have created a thioredoxin A deletion strain that shows decreased growth, an increased catalase activity, and the inability to form reproductive structures like conidiophores or cleistothecia when cultivated under standard conditions. However, addition of GSH at low concentrations led to the development of sexual cleistothecia, whereas high GSH levels resulted in the formation of asexual conidiophores. Furthermore, by applying the principle of thioredoxin-affinity chromatography we identified several novel putative targets of thioredoxin A, including a hypothetical protein with peroxidase activity and an aldehyde dehydrogenase.     

"Oxidative stress" response in submerged cultures of a recombinant Aspergillus niger (B1-D)

A recombinant strain of Aspergillus niger (B1-D), engineered to produce the marker protein hen egg white lysozyme, was investigated with regard to its susceptibility to "oxidative stress" in submerged culture in bioreactor systems. The culture response to oxidative stress, produced either by addition of exogenous hydrogen peroxide or by high-dissolved oxygen tensions, was examined in terms of the activities of two key defensive enzymes: catalase (CAT) and superoxide dismutase (SOD). Batch cultures in the bioreactor were generally found to have maximum specific activities of CAT and SOD (Umg x protein(-1)) in the stationary/early-decline phase. Continuous addition of H2O2 (16 mmole L(-1) h(-1)), starting in the early exponential phase, induced CAT but did not increase SOD significantly. Gassing an early exponential-phase culture with O2 enriched (25 vol%) air resulted in increased activities of both SOD and CAT relative to control processes gassed continuously with air, while gassing the culture with 25 vol% O2 enriched air throughout the experiment, although inducing a higher base level of enzyme activities, did not increase the maximum SOD activity obtained relative to control processes gassed continuously with air. The profile of the specific activity of SOD (U mg CDW(-1)) appeared to correlate with dissolved oxygen levels in processes where no H2O2 addition occurred. These findings indicate that it is unsound to use the term "oxidative stress" to encompass a stress response produced by addition of a chemical (H2O2) or by elevated dissolved oxygen levels because the response to each might be quite different.     

Expression of catalytic subunit of bovine enterokinase in the filamentous fungus Aspergillus niger

The cDNA encoding for catalytic subunit of bovine enterokinase (EK(L)), to which the sequence for Kex2 protease cleavage site was inserted, was expressed in the protease deficient filamentous fungus Aspergillus niger AB1.13. Fungal transformants were obtained in which expression of the glucoamylase fusion gene resulted in secretion of the protein into growth medium. Fusion polypeptide was processed to mature EK(L) by endogenous Kex-2 like protease cleavage during secretory pathway. The highest quantity of EK(L), up to 5 mg l(-1), was obtained in soya milk medium. The secreted EK(L) was easily purified from other proteins found in A. niger culture supernatant, using ion exchange and affinity chromatography. The yield of the purified and highly active EK(L) was 1.9 mg l(-1) of culture.     

Adaptive melanin response of the soil fungus Aspergillus niger to UV radiation stress at "Evolution Canyon", Mount Carmel, Israel

Background

Adaptation is an evolutionary process in which traits in a population are tailored by natural selection to better meet the challenges presented by the local environment. The major discussion relating to natural selection concerns the portraying of the cause and effect relationship between a presumably adaptive trait and selection agents generating it. Therefore, it is necessary to identify trait(s) that evolve in direct response to selection, enhancing the organism''s fitness. “Evolution Canyon” (EC) in Israel mirrors a microcosmic evolutionary system across life and is ideal to study natural selection and local adaptation under sharply, microclimatically divergent environments. The south-facing, tropical, sunny and xeric “African” slope (AS) receives 200%–800% higher solar radiation than the north-facing, temperate, shady and mesic “European” slope (ES), 200 meters apart. Thus, solar ultraviolet radiation (UVR) is a major selection agent in EC influencing the organism-environment interaction. Melanin is a trait postulated to have evolved for UV-screening in microorganisms. Here we investigate the cause and effect relationship between differential UVR on the opposing slopes of EC and the conidial melanin concentration of the filamentous soil fungus Aspergillus niger. We test the working hypothesis that the AS strains exhibit higher melanin content than strains from the ES resulting in higher UV resistance.

Methodology/Principal Findings

We measured conidial melanin concentration of 80 strains from the EC using a spectrophotometer. The results indicated that mean conidial melanin concentration of AS strains were threefold higher than ES strains and the former resisted UVA irradiation better than the latter. Comparisons of melanin in the conidia of A. niger strains from sunny and shady microniches on the predominantly sunny AS and predominantly shady ES indicated that shady conditions on the AS have no influence on the selection on melanin; in contrast, the sunny strains from the ES displayed higher melanin concentrations.

Conclusions/Significance

We conclude that melanin in A. niger is an adaptive trait against UVR generated by natural selection.     

Presence and regulation of the alpha-ketoglutarate dehydrogenase multienzyme complex in the filamentous fungus Aspergillus niger.

alpha-Ketoglutarate dehydrogenase has been demonstrated for the first time in cell extracts from the filamentous fungus Aspergillus niger. A minimum protein concentration of 5 mg/ml is necessary for detecting enzyme activity, but a maximum of ca. 0.060 mumol/min per mg of protein is observed only when the protein concentration is above 9 mg/ml. alpha-Ketoglutarate can partly stabilize the enzyme against dilution in the assay system. Neither bovine serum albumin nor a variety of substrates or effectors of the enzyme could stabilize the enzyme against inactivation by dilution. A kinetic analysis of the enzyme revealed Michaelis-Menten kinetics with respect to alpha-ketoglutarate, coenzyme A, and NAD. Thiamine PPi was required for maximal activity. NADH, oxaloacetate, succinate, and cis-aconitate were found to inhibit the enzyme; AMP was without effect. Monovalent cations including NH4+ were inhibitory at high concentrations (greater than 20 mM). The highest enzyme activity was found in rapidly growing mycelia (glucose-NH4+ or glucose-peptone medium). We discuss the possibility that citric acid accumulation is caused by oxaloacetate and NADH inhibition of the alpha-ketoglutarate dehydrogenase of A. niger.     

Characterisation of CwpA, a putative glycosylphosphatidylinositol-anchored cell wall mannoprotein in the filamentous fungus Aspergillus niger

Glycosylphosphatidylinositol (GPI)-anchored proteins in fungi are found at the cell surface, either as plasma membrane proteins (GPI-PMPs) or attached by a remnant of the GPI-anchor to the cell wall (GPI-CWPs). GPI-CWPs can be extracted from the cell wall by treatment with hydrofluoric acid (HF), which cleaves the phosphodiester bond that is present in the remnant of the GPI-anchor. The filamentous fungus Aspergillus niger contains at least seven HF-extractable cell wall mannoproteins. One gene encoding an HF-extractable cell wall mannoprotein, cwpA, was cloned and further characterised. The protein sequence of CwpA indicated the presence of two hydrophobic signal sequences both at the N-terminus and C-terminus of the protein, for entering the ER and the addition of a GPI-anchor, respectively. A CwpA-specific antiserum was raised and in combination with fractionation experiments, we show that this protein was abundantly present as an HF-extractable protein in the cell wall of A. niger.     

Transformation of the filamentous fungus Gibberella fujikuroi using the Aspergillus niger niaD gene encoding nitrate reductase

Summary A transformation system for Gibberella fujikuroi based on the Aspergillus niger nitrate reductase gene (niaD) was developed. A strain (designated SG140) carrying a non-reverting niaD mutation (niaD11) was generated by screening mutagenised cells for non-growth on nitrate as sole nitrogen source. Transformation frequencies of 1–2 transformants per g DNA were observed when strain SG140 was transformed to nitrate utilisation. Southern blot analyses of niaD⁺ transformants showed that the vector DNA sequences were integrated into the chromosomal DNA. The results demonstrate that the A. niger niaD gene is expressed in G. fujikuroi.     

Bioconversion of waste office paper to gluconic acid in a turbine blade reactor by the filamentous fungus Aspergillus niger

Gluconic acid production was investigated using an enzymatic hydrolysate of waste office automation paper in a culture of Aspergillus niger. In repeated batch cultures using flasks, saccharified solution medium (SM) did not show any inhibitory effects on gluconic acid production compared to glucose medium (GM). The average gluconic acid yields were 92% (SM) and 80% (GM). In repeated batch cultures using SM in a turbine blade reactor (TBR), the gluconic acid yields were 60% (SM) and 67% (GM) with 80-100 g/l of gluconic acid. When pure oxygen was supplied the production rate increased to four times higher than when supplying air. Remarkable differences in the morphology of A. niger and dry cell weight between SM and GM were observed. The difference in morphology may have caused a reduction of oxygen transfer, resulting in a decrease in gluconic acid production rate in SM.     

Cd (II) stress response during the growth of Aspergillus niger B 77

Aims:  To investigate the relationship between growth, heavy metal ions uptake and participation of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in the protection of Apergillus niger B 77 against cadmium stress.
Methods and Results:  The stress response of the model fungal strain, under conditions of a wide range of Cd (II) ion concentrations, was investigated by determining the biomass formation, protein biosynthesis, SOD and CAT activities and heavy metal uptake in growing cells. Exposure to heavy metal ions induced an increase in protein content, heavy metal uptake and SOD activity, and a heavy decrease in CAT activity.
Conclusion:  The results obtained indicated that the tolerance of A. niger to Cd (II) was correlated with the heavy metal uptake, reactive oxygen species generation in the cells and the efficiency of antioxidative defence system.
Significance and Impact of the Study:  Evidence is provided for the possibility that oxidative stress plays a major role in the effect of Cd (II) ions on A. niger . These data could offer useful information when creating new strategies and methodological improvements for bioremediation with the participation of fungi.     

Septum-directed secretion in the filamentous fungus Aspergillus oryzae

Although exocytosis in fungal cells takes place at hyphal tips, there also seems a line of circumstantial evidence suggesting the occurrence of exocytosis at other sites of cells, such as septa. To investigate whether exocytosis takes place at fungal septa, we monitored dynamics of EGFP‐fused α‐amylase (AmyB–EGFP), the representative secretory enzyme of the filamentous fungus Aspergillus oryzae. We found that AmyB–EGFP accumulates in Spitzenkörper at hyphal tips as well as septal periplasm between the plasma membrane and cell walls. The septal accumulation of AmyB–EGFP was a rapid process, and required microtubules but not F‐actin. Thus, this process is independent of exocytosis at hyphal tips that requires both microtubules and F‐actin. In addition, fluorescence recovery after photobleaching (FRAP) analysis of EGFP‐fused AoSnc1 revealed that secretory vesicles constitutively fuse with the septal plasma membrane. These results demonstrated that exocytosis takes place at septa in addition to hyphal tips. Analysis of two plasma membrane transporters, AoUapC and AoGap1, revealed that they preferentially accumulate at septa and the lateral plasma membrane with no clear accumulation at apical Spitzenkörper, suggesting that non‐tip directed exocytosis is important for delivery of these proteins.     

Osmotic adjustment in the filamentous fungus Aspergillus nidulans.

Aspergillus nidulans was shown to be xerotolerant, with optimal radial growth on basal medium amended with 0.5 M NaCl (osmotic potential [psi s] of medium, -3 MPa), 50% optimal growth on medium amended with 1.6 M NaCl (psi s of medium, -8.7 MPa), and little growth on medium amended with 3.4 M NaCl (psi s of medium, -21 MPa). The intracellular content of soluble carbohydrates and of selected cations was measured after growth on basal medium, on this medium osmotically amended with NaCl, KCl, glucose, or glycerol, and also after hyperosmotic and hypoosmotic transfer. The results implicate glycerol and erythritol as the major osmoregulatory solutes. They both accumulated during growth on osmotically amended media, as well as after hyperosmotic transfer, except on glycerol-amended media, in which erythritol did not accumulate. Furthermore, they both decreased in amount after hypoosmotic transfer. With the exception of glycerol, the extracellular osmotic solute did not accumulate intracellularly when mycelium was grown in osmotically amended media, but it accumulated after hyperosmotic transfer. It was concluded that the extracellular solute usually plays only a transient role in osmotic adaptation. The intracellular content of soluble carbohydrates and cations measured could reasonably account for the intracellular osmotic potential of mycelium growing on osmotically amended media.