CHICAT: Chitin catabolism in filamentous fungi

Chitin is a linear polysaccharide composed of ß-(1-4) linked residues of N-acetylglucosamine units. In filamentous fungi chitin is located in the inner layers of the cell wall, close to the plasma membrane and forms together with ß- (1,3) glucan the structural scaffold of the fungal cell wall. Chitin is in nature not only found in fungal cell walls, but also in the exoskeletons of protists and arthropods, e.g. insects and shrimps. Chitin does not visibly accumulate in the biosphere, which shows that the huge amounts of annually produced chitin - at least 10 gigatons - also are degraded each year. The main degraders of chitin are bacteria and fungi, which are able to use chitin as a nutrient source. In fungi, chitinases are not only important for external chitin degradation for nutritional purposes, but are also involved in internal chitin degradation during cell wall remodeling and recycling. Chitinases decompose the chitin polymer into shorter oligomers with a minimal chain length of 2 sugar subunits. This dimer needs to be converted into monomers by N-acetylglucosaminidases (chitobiases), before it is taken up into the fungal cell and metabolized. Interestingly, fungi have usually between 10 and 30 different chitinases, but only one or two N- acetylglucosaminidases. The further degradation and conversion of the monomer N-acetylglucosamine has so far only been studied in the human pathogenic yeast Candida albicans, but has not been investigated in filamentous fungi yet. We found that in filamentous fungi the genes involved in N-acetylglucosamine catabolism - in analogy to C. albicans - are clustered. However, in contrast to C. albicans, this gene cluster contains in filamentous fungi also a transcription factor and an enzyme belonging to glycoside hydrolase family 3. The aims of this project are to investigate the transcriptional regulation of this gene cluster in fungi of the genus Trichoderma, for which the extracellular chitinolytic enzyme machinery has already been well described. Biochemical characterization of the glycoside hydrolase family 3 protein will also show a potential role of this enzyme in N-acetylglucosamine catabolism. Further, the functions of the respective N-acetylglucosamine cluster genes - with focus on the transcription factor - will be characterized by generation of gene knockout strains in order to elucidate metabolic and regulatory roles of these proteins in chitin degradation in filamentous fungi.

Chitin is a linear biopolymer consisting of N-acetylglucosamine sugar units. In nature, it is found in the exoskeleton of insects and crustaceans as well as in the cell wall of fungi. In the biosphere, microorganisms, i.e. bacteria and fungi, naturally recycle it. They degrade chitin into its monomeric sugar units, which are subsequently taken up and used as nutrient and energy source. In this project, we investigated the metabolic pathways that are necessary for uptake and intracellular degradation of N-acetylglucosamine. Advancing our understanding of N-acetylglucosamine metabolism in fungi is not only relevant for investigations of natural chitin turnover in soil and marine habitats, but also for potential transfer of this knowledge to biotechnological processes that involve filamentous fungi in the valorization of fungal biomass. N-acetylglucosamine and chemically related compounds are important components in a wide range of products, .e.g. cosmetics, food supplements and pharmaceutical products. Analysis of genome sequences of filamentous fungi revealed that the genes for the N-acetylglucosamine degradation pathway are clustered in fungi. We could show in this project that they are co-regulated and under the control of a novel transcription factor, RON1. Our results clearly emphasized the essential role of RON1 for N-acetylglucosamine degradation and thus for the use of this sugar for nutritional purposes in fungi. Further, the function and gene regulation of the entire N-acetylglucosamine pathway was characterized. A comparison of various fungal species showed that they exhibit strong similarities on the genomic level with respect to the organization and genetic composition of the N-acetylglucosamine pathway, but growth tests interestingly revealed that this sugar can be strongly growth inhibiting for some fungal species. This was somewhat surprising because the respective fungi contain significant amounts of the very same sugar as polymer in their own cell walls. The reason for the strong growth defects is probably that one of the intermediate products of the N-acetylglucosamine degradation pathway is particularly toxic for these fungal species. These results emphasize that understanding such biological connections can be very important for the proper selection and adaptation of microorganisms for biotechnological processes.