Research

Wider research context: Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave glycosidic bonds in polysaccharides, thus boosting the activity of well-known hydrolytic depolymerizing enzymes. The process involves molecular oxygen/hydrogen peroxide and an electron donor, such as enzymes of GMC oxidoreductase family (e.g. cellobiose dehydrogenase), small-molecule reductants or photoactive pigments. Clonostachys rosea (Hypocreales, Ascomycota) is a filamentous fungus that colonizes living plants as an endophyte, and parasitizes on and kills other fungi (necrotrophic mycoparasite). Upon sequencing of the C. rosea genomes, the gene family encoding LPMOs (AA9), as well as the GMC oxidoreductase family (AA3) were found to be significantly expanded. In contrast, the genomes of the saprotrophic and mycoparasitic Trichoderma species (ecologically very similar to C. rosea) have significantly low number of AA9 and AA3 genes. Research objectives: We aim to unravel the catalytic activities and substrate specificities of C. rosea novel LPMOs, identify new structure-function aspects in combination with biochemical characterizations, and study their synergies with other native C. rosea enzymes such as GMC oxidoreductases.

The funds of the third BiMM funding period will be used to complete the ongoing work in the field of new bioactive substances and to establish an insect screening procedure and for “exploratory research”, which will then serve as the basis for further research applications to other funds. The research plan contains an “open part” for the topics ‘insect screening’ and “exploratory work” as well as a series of work packages for the more detailed characterization of the 5 candidates for new bioactive substances. One of them is BiMM20 (bimmycin), which comes from a new fungal species isolated in a BiMM project and is already well characterized. It has a fungicidal effect. One of the four other molecules has an antibacterial effect against Gram+ organisms; further research is being carried out into its mode of action. Another molecule can break the resistance of bacteria to chloramphenicol and thus restore sensitivity to this important antibiotic. Another molecule from a new, previously undescribed species of fungus from BiMM's own collection has a strong phytotoxic effect. The identity of this molecule and its properties are also being determined in this BiMM III project. The last of the five new molecules has already been tested in collaboration with the FH IMC Krems and found to have anti-inflammatory properties. Funding from the BiMM III project is also required to finance further characterization with a view to medical application. In addition to the specific studies on the new metabolites and their activities, the funds will also be used for exploratory research aimed at establishing new screening systems within BiMM. This includes, for example, the possibility of testing extracts and molecules for their insecticidal effect with a higher throughput.

Understanding the central switch in fungal toxin production Molds play a variety of ecological roles on our planet, especially in natural nutrient cycles and as pathogens of plants and animals. Of particular interest is their ability to produce a wide variety of small, bioactive molecules. One example of a very useful molecule is the antibiotic penicillin, which has saved millions of lives to date. But there are also dangerous molecules, such as toxins produced by molds, that can contaminate our food and thereby threaten consumer health. So the final goal must be to precisely understand the molecular and cellular programs in those fungi and thus develop natural solutions against fungal infections. Our previous research has shown that epigenetic processes regulate toxin production, but also a specific kinase - a regulatory protein that can chemically modify other proteins in its environment - plays a decisive role. In a previously unknown way, it is able to transmit a starvation signal in the cell, which ultimately leads to a complete change in the cellular metabolism and thereby activates the genetic machinery for toxin formation. In this project, we are now addressing the question of exactly how the starvation signal is processed by the fungal cell so that mycotoxins are formed. For this, we are using as experimental systems a conventional mold that is present ubiquitously in nature and buildings (Aspergillus) and a fungus that is considered one of the most important pathogens in crop plants worldwide (Fusarium). The entire genetic response of both fungi at the RNA level and at the protein level will be analyzed molecularly, biochemically and bioinformatically during different stages of their life cycles. In addition, those proteins whose function is regulated by the kinase should be identified. In this way, the signaling pathway from nutrient deficiency from the cell surface to the genetic activation of toxin production in two different molds should be elucidated as completely as possible. Ultimately, the results of this research could close a gap in the fundamental understanding of cellular processes during toxin production in mold fungi. This knowledge can then be used to develop new control strategies to ward off plant diseases and prevent biological contamination of food.