Abstract
Agaricus bisporus, the white button mushroom, is economically the most important mushroom cultivated worldwide. Growth of A. bisporus needs a substrate produced by the composting of animal manure, wheat straw, gypsum, water and different additives. Therefore lignocellulose which is a complex mixture of cellulose, hemicellulose and lignin usually constitutes an
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important fraction of the total organic matter. Degradation of plant biomass is an important aspect of the cultivation process of edible mushrooms, but the mechanisms behind it are still largely a black box. The commercial cultivation of A. bisporus offers a unique opportunity to study the interaction of a fungus with its substrate. In addition, each ton of produced mushrooms leaves almost the same amount of spent compost which contains approximately one third of carbohydrates from the original compost. Therefore the aim of this PhD thesis was to obtain a deeper understanding of how A. bisporus grows in compost and degrades its substrate, and to study the molecular mechanisms underlying this phenomenon. Multi-omics studies together with substrate composition analysis and measurements of enzymatic activities were performed to understand the unique mechanisms of lignocellulose degradation and conversion by A. bisporus. The genome sequence of A. bisporus identified a wide set of genes encoding plant cell wall Carbohydrate Active enZymes (CAZymes) and detailed analysis of the expression of these genes together with genes involved in central carbon metabolism was performed in compost, casing layer and fruiting bodies of the first flush. To further evaluate degradation of the substrate by A. bisporus in-depth integrative analysis of transcript expression and protein secretion as well as carbohydrate degrading enzyme assays data from A. bisporus mycelium grown compost at different growth stages was performed. To make transcriptome analysis possible, the method based on the CsCl gradient ultracentrifugation was developed and allowed obtaining high levels of quality and quantity of RNA. Overall, the analysis of the genome and transcriptome of A. bisporus revealedthe unique repertoire of genes and its expression pattern showed the adaptation of this fungus to humic rich partially decomposed leaf material. Detailed analysis of the genes encoding plant and fungal polysaccharide modifying enzymes and central carbon metabolism demonstrated differences in these genes expression between compost- casing layer-grown mycelium and fruiting bodies. Further investigations of transcriptome and proteome for abilities of A. bisporus to degrade plant biomass throughout its life cycle showed changes in gene expression involved in (hemi-)cellulose degradation between the first and second flushes could partially explain the reduction in the yield of mushrooms during the second flush. Moreover, exploring unutilized fractions which remain in compost, arabinosyl and glucuronic acid substituents of the xylan backbone were found to be accumulated in the compost towards the end of the mushroom cultivation. These observations may explain the plant biomass degrading abilities of A. bisporus, revealing a set of potential important genes as well as secreted enzymes, including those which are missing and potentially significant for the substrate degradation.
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