The complex sugars ('polysaccharides') found in the cell-walls of plants impact on all our everyday lives. They form the cotton of our clothes, the physical origins of paper and are central to all the, many, industries that derive from these products. Plant cell wall sugars are likely to form the basis of the next generation biofuel industry; one of the key governmental strategies to provide us with green, renewable and secure energy for the twenty-first century. Furthermore, the harnessing of recalcitrant sugars ('roughage') by our gut microbes is an essential part of human nutrition and intestinal transit. The gut flora are inextricably linked to human health through their metabolism of complex plant polysaccharides. The aim of this BBSRC grant application is to study how nature's catalysts, termed 'enzymes', digest and breakdown plant polysaccharides. These catalysts are the vanguard of the industries that are springing-up with the goal of harnessing the plant cell wall. Enzymes are the actors on the complex stage of the human intestine. The application focuses mainly, on one particularly difficult class of sugars; the xyloglucans. We aim to show how complex enzyme systems are required for the breakdown of this polysaccharide, how these enzymes function both at the three dimensional level and how they can be used as catalysts with potential industrial applications. We will study both key, selected representative enzymes showing useful properties and whole enzyme systems from human gut bacteria. We hope to provide the community with insight into the function and action of these catalysts and provide a foundation or their exploitation to aid in human health and technology development.

Technical Abstract:
The plant cell wall is the biggest reservoir of organic carbon in the biosphere. Fixed carbon dioxide is present in numerous plant polysaccharides notably cellulose and the diverse 'hemicellluloses' including xylan and xyloglucan (XG). The industrial exploitation, through targeted enzymatic hydrolysis, of the plant cell wall is central to many industries. Historically this includes the pulp and paper, textile and detergent sectors but more recently the drive for renewable, energy-secure and green alternatives to fossil fuel depletion has raised the importance of enzymatic biomass conversion for biofuels. Here, we target the diverse xyloglucanases that are responsible for the removal of recalcitrant (where they 'stick' to cellulose rendering enzymatic hydrolysis difficult; highlighted by NREL as a major stumbling block in biofuel projects) XGs from plant biomass. In preliminary work we have cloned, expressed and crystallised (diffraction >2Å) a number of key enzymes responsible (a-xylosidases and their relatives) as well as unusual XGases that target the previously intractable 'xylose-linked' glucose in the XG backbone. Furthermore, we have applied a systems-approach to identify a XG degrading system from Bacteroides ovatus in the human gut. This human symbiont displays a remarkable XG 'polysaccharide utilization operon (PUL)' contain the full repertoire of enzymes required for the degradation of dietary XGs (XGs constitute up to 20% of the dry mass of dietary plants such as salad and tomato) as well as providing key enzyme classes currently absent from the classical industrial repertoire such as those require to 'de-arabinosylate' XGs. Many of these XG-PUL genes have been expressed, in E coli, at high levels. We will dissect these numerous XG degradation systems, determine the three-dimensional structural foundation for enzyme action, define the kinetic and mechanistic basis of catalysis and provide a panel of novel enzymes for the biofuel and related industries.

Potential Impact:
Who will benefit from this research? The degradation of plant-derived biomass is one of the key objectives of BBSRC policy, UK government policy and of many UK and European Biotechnology companies. Therefore the spectrum of 'stakeholders' in this work is very large indeed. UK biotechnology, albeit belatedly compared to Scandinavian enterprises, is embarked on major expansions in the 'enzymatic treatment of plant polysaccharide' sector. Enzyme such as those described in the application find everyday application that touches on the general public in their paper and packaging products, in household detergents and washing products, in household foodstuffs such as sugar (derived by enzymatic treatment of corn-starch). These 'day-to-day' benefits of the wider public are in addition to the potential massive benefits of reducing fossil fuel usage, allowing greener industries, and fuel security as part of a balanced UK energy portfolio. How will they benefit from this research? It is clear that information derived from this project will provide UK and European industry with key enzymes, and a basis for their engineering and exploitation, in the plant polysaccharide sector. This is a well trodden 'route' for the biotechnology sector (enzyme isolation - enzyme characterization - 3D structure - engineering and optimisation in industry for specific consumer benefit). What the work described in the application provides is a new enzyme portfolio and the release into the public sector of sequences, knowledge of activities and 3-D structures upon which enzyme optimisation will be based. In particular this knowledge is in an area 'hemi-cellulose degradation' that the key industrial leaders and US National Renewable Energy Laboratory has highlighted as a major bottleneck in their applications. The application of new enzymes goes far wider, however, than biofuels. Plant polysaccharidases find numerous uses in paper & pulp, food and textile industries with applications from green enzymatic synthesis of novel oligo-and polysaccharides, through cosmetic delivery, washing-powder additives, animal feed pre-treatment and the synthesis of 'designer' paper products (currently a major initiative in both Scandinavia and Canada). Results from this grant will be in the public domain within months of publication submission. Industry and policy leaders will therefore have almost immediate access to raw data. Staff Training Staff employed on this project, and any BBSRC-funded students they help supervise, will clearly have skills directly relevant to one of the growing sectors of UK, and worldwide industries. Furthermore, the enzyme mechanisms of plant-degrading enzyme systems, and the skills / techniques used to study them, are VERY similar to those used in the drug industry to target enzymes. Flu virus neuraminidase, for example, is a sugar hydrolase using similar mechanisms to the plant degradation apparatus. Thus staff employed on this project will equally well be appropriately trained to enter into the UK pharmaceutical industry.