Enzyme action and hemicellulose substrate heterogeneity in plant cell walls

The project will use complementary approaches to dissect enzyme action on isolated cell wall polysaccharides and intact cell walls from Miscanthus sepcies. The project is a collaboration with Prof. Simon McQueen-Mason at the University of York. The Leeds group will use novel assays to assess the heterogeneity of Miscanthus cell walls and to explore this structural heterogeneity and how enzymes can act upon hemicellulose substrates in cell wall contexts. This will involve the use of extended sets of monoclonal antibody (MAB) and carbohydrate-binding module (CBM) probes directed to cell wall polysaccharides including probes for xylan backbones in addition to arabinosyl, glucuronosyl, and feruloyl substitutions. At York complementary novel high throughput quantitative assays for Miscanthus cell wall degradability and saccharification will be used to monitor the capacities for deconstruction of the cell wall materials. These technologies and approaches are also suitable for high throughput screening of enzymes. The project will systematically study the capacities of sets of enzymes and enzyme/CBM constructs produced in E. coli and (provided by other partners of the network) to target and degrade Miscanthus cell wall polymers using quantitative saccharification assays (York) and also in situ fluorescence imaging and microscale quantitative assays using sets of MAB and CBM probe-based assays (Leeds). The project will develop a clear understanding of both the heterogeneity of Miscanthus cell wall polysaccharides and its occurrence in the context of cell wall architecture in terms of polysaccharide associations and how this influences enzyme access to substrates and enzyme efficacy. The project will be in collaboration with research teams designing and engineering versions of the key enzymes characterised in this project to create an E. coli that can make the degradation enzymes as well as use all the products.