Hydrolase-Screening in Secretomes of Thermophilic Fungi

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Mini symposium: Glycobiology at the LUMC and beyond

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Hydrolase-Screening in Secretomes of Thermophilic Fungi

Geronimo Heilmann 1 , Leonard Sewald 1 , Christian Schmerling 2 , Bettina Siebers 2 , Markus Kaiser 1

1 University of Duisburg-Essen, Chemical Biology, Essen, Germany, 2 University of Duisburg-Essen, Molecular Enzyme Technology and Biochemistry, Essen, Germany

Fungi are a unique kingdom within the eukaryotic domain. Its subtaxa are highly diverse but all fungi share common features such as a distinct cell wall morphology and specific unique metabolic pathways. Among them, thermophilic fungi are particularly interesting because they are able to metabolize nutrients that are inaccessible for other eukaryotes under higher temperature conditions. For example, thermophilic fungi such as Phanerochaete chrysosporium are able to degrade wood and other non-food biomass and thus exhibit and secrete a huge repertoire of specific enzymes with potential applicability in biotechnology. [1, 2]

Here we show that Activity-based Protein Profiling (ABPP) is a promising method for functional screening and identifying hydrolases of thermophilic fungi. To this end, different hydrolase probes were used for in vivo screening and protein targets were identified by LC MS/MS.

P. chrysosporium cultures were chemically profiled under different nutrient conditions with a fluorophosphonate-based probe and detected a nutrient-dependent serine hydrolase repertoire. We identified three esterases, which hydrolyze chemical modifications on hemicellulose. These esterases catalyze the first step of (hemi)cellulose degradation by mediating access to the polymers for glycosidases. Moreover, we could identified several glycosidases in the same experiment. Recent work suggests that secreted esterases and glycosidases form synergistic protein complexes (Cellulosome) for targeted (hemi)cellulolytic activity.[3,4] Further studies to extend this approach to other, biotechnological relevant enzymes are currently underway.

Wymelenberg, A. V., Minges, P., Sabat, G., Martinez, D., Aerts, A., Salamov, A., ... & Dosoretz, C. (2006). Computational analysis of the Phanerochaete chrysosporium v2. 0 genome database and mass spectrometry identification of peptides in ligninolytic cultures reveal complex mixtures of secreted proteins. Fungal Genetics and Biology, 43(5), 343-356.

Martinez, D., Larrondo, L. F., Putnam, N., Gelpke, M. D. S., Huang, K., Chapman, J., ... & Coutinho, P. M. (2004). Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nature biotechnology, 22(6), 695.

Biely, P., Puls, J., & Schneider, H. (1985). Acetyl xylan esterases in fungal cellulolytic systems. Febs Letters, 186(1), 80-84.

Haitjema, C. H., Gilmore, S. P., Henske, J. K., Solomon, K. V., De Groot, R., Kuo, A., ... & Chiniquy, J. (2017). A parts list for fungal cellulosomes revealed by comparative genomics. Nature microbiology, 2(8), 17087.

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