Regulation of overflow metabolism in Penicillium

Filamentous fungi have an enormous ecological, biotechnological, agricultural and medical impact in our world. The physiology of these fungi is, however, only poorly understood in many respects. One approach to a better understanding is to gain highly time-resolved, quantitative data on the dynamic interactions between different levels of energy metabolism. This is, however, especially difficult with these fungi due to their highly environment-sensitive phenotypic plasticity (i. e. the rapid change in composition, morphology and physiology, when the environmental conditions vary. To follow this phenotypic plasticity requires an unusual high degree of standardization of cultivation conditions. This project aims at simultaneously gaining, data from five different levels of energy metabolism: overflow metabolism (organic acid excretion), the plasma membrane (membrane transport is a main energy consumer in the cell), as well as the energy charge, theredox status, and respiration. The necessary high degree of standardization of cultivation conditions will be achieved by using a chemostat, as well as automated highly time-resolved data acquisition of cultivation parameters during bioreactor batch cultivation. The data are gained from our model fungus, Penicillium ochrochloron, a heavy metal tolerant soil fungus, which can be used for the winning of metals from industrial wastes and low-grade ores. The dynamics of these levels of energy metabolism will be studied (i) with different nutrient limitations at constant environmental conditions in the chemostat, (ii) after the relief of a nutrient limitation, and (iii) after the onset of a nutrient limitation. Beyond this project the development of a high degree of standardization concerning growth phase determination in bioreactor batch culture will help to improve proteomics and metabolomics with filamentous fungi in general. Additionally, this systems biology approach will facilitate the modelling and prediction of the behaviour of filamentous fungi to changing environmental conditions. This is particularly valuable for biotechnological and medical applications. Three national and one international co-operation partner will help to tackle this task: Prof. Markus Ganzera (Institute of Pharmacy, Innsbruck), Prof. Andrey Kuznetsov (Daniel Swarovski Research Laboratory, Innsbruck), Prof. Günther Daum (Institute of Biochemistry, Graz), as well as Prof. Christer Larsson (Center for Molecular Protein Science, Lund, Sweden) and Prof. Nestor Torres (Institute for Biochemistry and Molecular Biology, La Laguna, Tenerife, Spain.)

Using a model organism for filamentous fungi the heavy metal resistant soil fungus Penicillium ochrochloron, a close relative of the penicillin producing Penicillium chrysogenum first it was shown that adaptations of metabolism to environmental and growth conditions are specific for the nutrient limitation and the growth phase. Second, it was further established that methods for physiological analysis and subsequently also for systems biology studies (e. g. sampling, stop of metabolism, sample preparation, metabolite extraction, metabolite analytics) must be adapted to the specific physiological state much more strongly as supposed up to now to attain reproducible and meaningful results. And third, for the first time in a filamentous fungus data were acquired which interrelate four levels of energy metabolism. These findings are significant to understand ecosystems in which fungi live, but all the more for biotechnological processes using filamentous fungi.Filamentous fungi, e. g. moulds, must adapt their metabolism both in nature and in biotechnological processes to different and rapidly changing environmental conditions (in contrast to animal cells which are provided a constant milieu). The strategies fungal cells (hyphae) use for the utilization of the main nutrient glucose was the core theme of this project. The amount of energy extracted from glucose must be matched to the available amount of other nutrients. Depending on what nutrient is limiting growth, this adaptation occurs at different levels of metabolism and with different mechanisms. We studied the respiration rate (oxygen consumption), the conversion rate in the cellular energy currency ATP, the activity of the main ATP consuming enzyme in the plasma membrane, and the excretion of metabolites, which is a kind of safety valve if glucose is in excess, but growth is limited by one or more of other nutrients.Without filamentous fungi most ecosystems would not be functioning from soils to oceans, from the tropics to arctic areas. In biotechnology specific species serve as source and cell factories for producing enzymes, versatile applicable substances such as citric acid, and medicinal relevant substances, for instance antibiotics. On the other hand some of these fungi cause large damages to agricultural cultures and spoil crops. The results of this project contribute to a better understanding of the energy metabolism of this eminent group of organisms, which have much to offer for a more sustainable way of human life.