To achieve the goal of producing biofuel from plant biomass (lignocellulose), the plant cell wall can be degraded by a cocktail of hydrolase enzymes that generate monosaccharide sugars (saccharification). Biomass feedstocks are pretreated to increase enzyme accessibility of the cellulose and hemicelluloses prior to addition of the enzyme cocktails. The released sugars are then industrially fermented to generate biofuels such as ethanol and butanol. The viability of lignocellulosic biofuel technology will depend on maximising the fermentable sugar from biomass, and minimising the costs of processing. Currently, it is difficult to use the pentose-rich hemicellulose xylan component which constitutes 20- 30% of most feedstocks such as grass and wood. This xylan impedes enzyme access to the cellulose, in part through links with the lignin. One of the main problems is that it is a branched polymer that is difficult to break down with enzymes. Acid treatments to break up the hemicellulose can generate inhibitors that prevent effective microbial fermentation and reduce the yield of sugars. This programme aims to achieve a better understanding of the genetic control of hemicellulose synthesis, especially the branched xylan component of biomass, and the impact of xylan branching on enzyme accessibility. It will develop a comprehensive characterisation of plant polysaccharide synthesis machinery, and how the synthesis enzymes work together in protein complexes. The programme will also discover and characterise effective enzymes that break down this component to monosaccharides. The programme will deliver enabling technologies for high throughput, detailed, quantitative analysis of biomass hemicelluloses and the activity of the enzymes that break them down. Based on this knowledge, strategies of plant breeding or modification, and also of hydrolytic enzyme selection, will be proposed in order to reduce the costs of use of the branched xylan component of biomass, and to release the cellulose for saccharification. The programme in Cambridge to study cell wall synthesis and to develop the polysaccharide and hydrolase profiling technologies is supported by enzyme discovery in the University of Newcastle, with Dr David Bolam and collaboration with Professor Harry Gilbert. Shell Global Solutions are collaborators in the programme, providing an important industrial perspective and bioinformatic support. Additional enzymes for method development and for analysis of cell wall polysaccharides will be provided and studied in collaboration with Novozymes, the world leader in enzyme production.

Technical Abstract:
1. We will develop enabling technologies for polysaccharide analysis. The field does not have the methods for high resolution screening, at the level of individual polysaccharide structures, of large populations of plants for natural or induced variation in polysaccharide quality and quantity. We also need to be able to study more precisely the specificity of hydrolytic enzyme actions. Our current techniques of PACE and LC-MS will be extended in scope, robustness and speed in this programme. 2. We will study hemicellulose degradation pathways and novel hydrolytic enzyme action. Enzymes required to remove effectively the extensive sugar decorations on branched xylan are largely unknown. We will discover and study novel debranching enzyme action, assisted by using the new profiling technologies. 3. We will use a systems approach to understanding control of polysaccharide quantity and quality. We will integrate transcriptomic and proteomic data on Golgi polysaccharide synthesis proteins to discover the enzymes, pathways and control of branched xylan and other polysaccharide synthesis. By studying polysaccharides in multiple mutants in a high throughput fashion, using our profiling technologies, we will achieve a broader understanding of polysaccharide function and the control of synthesis. 4. We will assess the consequences, for depolymerisation by the various enzymes, of altering the biosynthesis of specific branched xylan structures. 5. Using the knowledge of biosynthetic pathways, polysaccharide structure and hydrolytic enzyme action, we will propose how to select or generate crop plants with the structure of the branched xylan optimised for maximal potential sugar yield with minimal enzyme input, to ensure maximal degradation and utilisation of the biomass.