The 16th European Conference on Fungal Genetics was held last month, and Vera Meyer and Arthur Ram, editors of Fungal Biology and Biotechnology, as well as students and postdocs from their labs, attended and selected their top 5 posters from the conference. We present a brief overview of these researchers and their research.
Katherina Garcia Vanegas
Postdoc, Department of Biotechnology and Biomedicine, Section for Synthetic Biology, Eukaryotic Molecular Cell Biology, DTU
A versatile high-throughput friendly system for construction and validation of fungal cell factories
Fungal production is expected to constitute a foundation stone in a future sustainable and green bio-economy; and fungal cell factories are already significant producers of industrial enzymes, pharmaceuticals, fuels, and food additives. However, it can be difficult, expensive, and time-consuming to construct and validate fungal factories. Our goal is to develop simple and efficient methods to manipulate fungal genome for this purpose. Here we demonstrate that the natural DNA repair machinery of fungi can quickly and inexpensively assemble complex DNA constructs from simple DNA fragments. Specifically, we demonstrate that 2-6 fragments can easily be fused together to form fungal plasmids, to introduce gene deletions, or to insert new genes. The simplicity of the method promises to cut away weeks of strain construction work and can easily be implemented into automated strain construction setups. Importantly, we demonstrate that the technology works in several fungi indicating that it may applied in many different species. Indeed, we have used the technology in our multi-species cell factory platform, DIVERSIFY, which allows construction of several fungal species simultaneously using the same building blocks. In this setup, the best species for production of a given compound can be quickly identified and used as a starting point for the development of a future fungal cell factory.
Juan Carlos Nunez-Rodriguez
Institute for Research in Biomedicine (IRB Barcelona) and Barcelona Supercomputing Center (BSC-CNS)
Invasive fungal diseases are an emerging global issue. In recent years resistance to multiple antifungal drugs is increasingly reported and treatments in humans are very limited. Candida glabrata is one of the most common human pathogenic fungi and is characterized by its ability to acquire resistance. We pondered upon whether the genomic and phenotypic adaptations that occur during this process have any trade-offs.
To address this question, we analyzed a large collection of resistant strains against different stressors. To quantitatively analyze the growth of all strains and their replicates in each condition, we developed an in-house large-scale phenotyping methodology in solid media named Q-PHAST. The results of phenotyping over 3500 spots were strikingly clear, 98% of the strains had some trade-off in response to stress. We are now studying how drug resistance is related to these trade-offs and whether we can use these vulnerabilities as targets for novel treatments. This journey between basic and translational science is exciting and we hope to share our findings soon!
University of Szeged, Faculty of Science and Informatics, Department of Biotechnology, Szeged, Hungary
Dual mode of action of Neosartorya (Aspergillus) fischeri antifungal protein 2 (NFAP2) on Candida albicans
Candidiasis is a fungal infection caused by a yeast belonging to the genus Candida. This type of infections can affect the whole body from the skin to the inner organs, and can be fatal in lack of an effective therapy. Nowadays, candidiasis represents a worldwide problem in the consequence of the increasing number of antifungal drug-resistant strains. These strains often show resistance to more than two types of antifungal medicines. Therefore, there is an urgent need to introduce new antifungal compounds in the anti-Candida therapy, which differ in their mode of actions from the conventional ones. Interestingly, the other types of fungi, the moulds, provide a potential solution to overcome this problem. Moulds are well-known as producers of antibiotics (for example, penicillin) for decades, but recently it was observed that they can secrete so-called antifungal proteins to their environment. These proteins are highly stable, and able to inhibit the growth of other fungi or kill them irrecoverably.
A good example for antifungal proteins is the NFAP2 secreted by the mould Aspergillus fischeri. In the last few years, some studies already reported its high antifungal efficacy against drug-resistant Candida strains, and its safe applicability in the treatment of drug-resistant superficial infection. However, its antifungal mechanism was unclear until now. Revealing the mode of action promotes the introduction of NFAP2 as a potential therapeutic drug. NFAP2 shows a concentration-dependent dual mode of action. It means, if NFAP2 is applied at high concentration it kills Candida cells by disrupting the plasma membrane within minutes. While, if NFAP2 is applied at lower concentration, it is taken up by the Candida cells, and inside the cell it inhibits the glycolysis. Both mechanisms result in cell death. Plasma membrane integrity is essential for yeast cell viability and normal function. Computational model indicated that NFAP2 interacts with inositol phosphorylceramides, substantial and specific components of biological membranes in yeasts. This interaction makes the membrane disruption effect to be fungal-specific. Glycolysis is a metabolic reaction which extracts energy from glucose. NFAP2 binds selectively to transketolase and malate dehydrogenase, the key enzymes of this ancient metabolic pathway, and inhibits their function. This leads to starvation and depletion of energy, and as a consequence, inhibition of cell multiplication and death finally. This dual mode of action renders NFAP2 to be a promising candidate to fight against drug-resistant Candida strains. Investigation the ability of yeasts to develop potent resistance mechanisms to NFAP2 is in progress.
Research group Biochemistry, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, Vienna, Austria
Austrian Centre of Industrial Biotechnology (ACIB GmbH), Muthgasse 18, Vienna, Austria
Citric acid is one of the most important bulk chemicals which is used in our everyday life. It is for example applied for acidifying and preserving our food and serves as a cleaning agent in our detergents. For about 100 years citric acid is not extracted from lemons but is biotechnologically produced by the conversion of sugar using the black mold Aspergillus niger. This process is well-studied, however, open questions still remain. One important aspect that is still not understood is the impact of manganese on the citric acid production process. In our work, we are investigating the regulatory mechanisms of manganese within the citric acid production process of A. niger and we found that the gene cexA, which is necessary for the secretion of citric acid, is negatively regulated by manganese. Now, our focus lies on further resolving the regulatory mechanism manganese has within the citric acid production process.
Victor M Gonzalez
University of Helsinki, Department of Microbiology
Utilization of CRISPR/Cas9-based methodology for genetic manipulation of the basidiomycete white-rot fungus Dichomitus squalens
The basidiomycete white-rot fungus Dichomitus squalens is an efficient wood-degrading species which produces a highly adjusted enzymatic response for degradation of various types of plant biomass. To study molecular mechanisms behind this process, functional characterization of plant biomass degradation related genes is needed, and this requires efficient and reliable genetic manipulation tools. While CRISPR/Cas9 tools have revolutionized genetic studies in several organisms including ascomycete fungi, tools for basidiomycetes are underdeveloped in comparison.
We have established a CRISPR/Cas9-based methodology in D. squalens, which enables precise genome editing for gene disruption. We have successfully used this methodology to construct D. squalens disruption mutants of candidate regulators controlling production of plant biomass degrading enzymes. The phenotypical characterization of these mutants has revealed essential regulators of plant biomass degradation.