Mining microbial compost communities for lignocellulose degrading proteins

The production of second generation biofuels from agricultural residues is an attractive alternative to the use of conventional first generation feedstocks, which are also important food resources. However, these alternative feedstocks predominately consist of lignocellulose, the main structural component of the plant cell wall, and expensive physicochemical and enzymatic pre-treatments are required before fermentation into biofuel. Therefore, the discovery of novel enzymes capable of deconstructing lignocellulose, in conditions that would be amenable to industry, is an important goal. The work, presented in this thesis, has explored the degradation of lignocellulose by a community of composting microbes, enriched for growth on wheat straw. Culturable members of the community, were isolated and assessed for their enzymatic activities towards lignin, cellulose and xylan. From these studies, a promising Ascomycota was identified, Graphium sp., which was capable of utilising both crystalline cellulose and xylan as carbon sources for growth. Transcriptomic studies were performed on Graphium sp. with and without wheat straw present, representing the first molecular information generated from an organism of this genus. From this 680 putative proteins were annotated as containing carbohydrate active domains. Proteomics added further depth to the analysis, with investigations focused on secreted proteins both located in the culture supernatant and bound to the insoluble lignocellulose substrate. Six secreted proteins were identified as targets for further analysis, and three of these were successfully isolated either from the native host, or a heterologous system. This included a lytic polysaccharide monooxygenase that appeared active on both chitin and cellulose, and a GH7 whose activity on cellulose was demonstrated. An intriguing protein, which showed low homology to a dioxygenase, was also expressed, though its role in the lignocellulose degrading environment has yet to be established.