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Although host proteases are often considered to have a negative impact upon heterologous protein production by filamentous fungi, relatively little is known about the pattern of their appearance in recombinant fungal bioprocesses. In the present study, we investigated extracellular proteases from a filamentous fungus, Aspergillus niger B1-D, genetically modified to secrete hen egg white lysozyme (HEWL). Our findings indicate that extracellular protease activity is only detected after the carbon source is completely utilised in batch cultures. The proteases are predominantly acid proteases and have optimal temperature for activity at around 45°C. Their activity could be partially inhibited by protease inhibitors, indicating the existence of at least four kinds of proteases in these culture fluids, aspartic-, serine-, cysteine-, and metallo-proteases. Oxygen enrichment does not have any noticeable effects on extracellular protease activity except that the onset of protease activity appears earlier in oxygen enrichment runs. Oxygen enrichment stimulates HEWL production substantially, and we propose that it is related to fungal morphology. Thermal stress imposed by raising process temperature (from 25 to 30 and 35°C) in early exponential phase, led to appearance of protease activity in the medium following the heat shock. Continued cultivation at high temperatures significantly reduced HEWL production, which was associated with increased activity of the extracellular proteases in these cultures. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, we used a recombinant filamentous fungus strain, Aspergillus niger B1-D, as a model system, and investigated the antioxidant defences in this organism. Our findings indicate that pretreatment with low concentrations of H2O2 completely prevents killing by this oxidant at high concentrations. It shows that A. niger adapts to exposure to H2O2 by reducing growth and inducing a number of antioxidant enzyme activities, including superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, of which the induction of catalase is the most pronounced. Moreover the decline of these antioxidant enzymes activities after H2O2 detoxification, coincides with recommencement of growth. Results from monitoring the extracellular H2O2 concentration clearly indicate a very rapid detoxification rate for H2O2 in adapted A. niger cultures. A mathematical model predicts only very low concentrations of intracellular H2O2 accumulating in such cultures. Our results also show that glutathione plays a role in the oxidative defence against H2O2 in A. niger. On addition of H2O2, the intracellular pool of glutathione increases while the redox state of glutathione becomes more oxidized. [Copyright &y& Elsevier]

Abstract: Dissolved carbon dioxide is one variable that changes between scales of bioreactor and may thus have a potential influence on the process as scale alters, and especially so in filamentous fungal cultures. The effects of dCO2 on growth and penicillin production by an industrial strain of Penicillium chrysogenum were examined systematically using a steam sterilisable dCO2 probe and a computerized image analysis system. Although low levels of CO2 stimulated growth (increased biomass, lower mean hyphal growth unit (M.H.G.U.) in lag phase) at the expense of antibiotic synthesis, higher levels led to elevated M.H.G.U.''s in the lag phase and were associated with increased clumping and severely reduced penicillin titre. The adverse effects of dCO2 in these cultures were largely mediated by inhibition of growth and substrate uptake in the lag phase, causing morphological changes which might also contribute to reduced penicillin levels. [Copyright &y& Elsevier]

Fungal autolysis is the natural process of self-digestion of aged hyphal cultures, occurring as a result of hydrolase activity, causing vacuolation and disruption of organelle and cell wall structure. Previously, authors have considered individual aspects of fungal lysis, in terms of either an enzyme, a process or an organism. This review considers both the physiology and morphology of fungal autolysis, with an emphasis on correlations between enzymological profiles and the morphological changes occurring during culture degeneration. The involvement of the main groups of autolytic hydrolases is examined (i.e., proteases, glucanases, and chitinases), in addition to the effects of autolysis on the morphology and products of industrial bioprocesses. We call for a concerted approach to the study of autolysis, as this will be fundamental for research to progress in this field. Increased understanding will allow for greater control of the prevention, or induction of fungal autolysis. Such advances will be applicable in the development of antifungal medicines and enable increased productivity and yields in industrial bioprocesses. Using paradigms in existing model systems, including mammalian cell death and aging in yeast, areas for future study are suggested in order to advance the study of fungal cell death. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: It is now accepted that heat treatment can lead to oxidative stress events in microorganisms, but there are few detailed studies on protein oxidation and consequent recycling/elimination of oxidatively damaged proteins following such heat treatment. In batch cultures of a filamentous fungus, Aspergillus niger B1-D, raising culture temperature from 25°C to 30°C and 35°C led to a general enhancement of intracellular metabolism, a higher specific growth rate, increased consumption of carbon and nitrogen sources, and raised intracellular ATP content in the exponential phase. By contrast, there was a transient accumulation of protein carbonyls, a widely used biomarker of protein oxidation, following such temperature increases, which could indicate that cellular antioxidant defences were being temporarily overwhelmed under these circumstances, despite the fact that enhanced activities in antioxidant enzyme activities have been reported in microorganisms during such heat treatment. Protein carbonyls can only be removed by proteolysis. The intracellular proteolytic activity in batch cultures of A. niger was found generally enhanced by temperature elevation, suggesting the role of proteolytic activity in protein quality control during heat treatment was dependant on the culture temperature. [Copyright &y& Elsevier]

Abstract: Filamentous fungi are industrially important organisms because of their ability to produce secondary metabolites, and as expression systems for heterologous proteins. Since secretion of secondary metabolites in filamentous fungi is associated with the older, more vacuolated parts of the hyphae culture morphology is a crucial reflection of their physiology in submerged cultures. Therefore, any factor affecting fungal morphology will in turn affect secondary metabolite production. In this study we examine and report the effects of elevated levels of the potent morphogen, carbon dioxide upon morphology and antibiotic production in an industrially important fungal system. The effects of elevated CO2 levels on cephalosporin C (CPC) production, metabolic performance and morphological changes in batch cultures of Acremonium chrysogenum (ATCC 48272) were investigated. High levels of dCO2 led to reduction of CPC production by up to 36%. Both metabolic activity and substrate uptake were severely inhibited by elevated CO2, especially at 15 and 20% CO2. Both micro and macromorphology of A. chrysogenum were profoundly changed in bioprocesses sparged with CO2 especially at high levels, and these changes were a contributory cause of the reduction in CPC production. [Copyright &y& Elsevier]

Abstract: Oxidative stress events have been shown to be associated with reduced consumption of nutrients in yeasts, but there are very few studies in filamentous fungi. In the present study we investigated the impact of oxidative stress on glucose and ammonia utilization in batch cultures of Aspergillus niger B1-D. The addition of 1mm H2O2 significantly reduced both glucose and ammonia uptake rates in these cultures. Associated with the decreased nutrient uptake, the activity of glyceraldehyde-3-phosphate dehydrogenase was greatly reduced; conversely, the activity of glucose-6-phosphate dehydrogenase remained unchanged. During the period of reduced nutrient uptake, the intracellular ATP and NADPH levels decreased while the amount of trehalose increased. The activities of glutamine synthetase and glutamate dehydrogenase, two key enzymes of ammonia assimilation, remained unchanged in response to H2O2 up to 1mm, suggesting the decreased ammonia uptake rate noted under such conditions is not due to enzyme inactivation caused by oxidative stress, but may be due to an insufficient supply of ATP and NADPH, which are required for ammonia assimilation. [Copyright &y& Elsevier]