Lignocellulosic biomass represents the greatest reserve of fixed carbon in the biosphere and its degradation is an important component of the global carbon cycle. Lignocellulose, in the form of crop residues and the biological fraction of municipal solid waste, also represents a potential low-carbon replacement for petroleum for the production of sustainable fuels, chemicals and materials. Lignocellulose is a rich potential source of sugars and phenolics as it is primarily comprised of polysaccharides and lignin. These can serve as a source of dietary nutrition for organisms that feed on it, or as platform chemicals for industry, but in both cases, obtaining these compounds is challenging due to the recalcitrance of lignocellulose. We are studying the digestive systems of invertebrate animals specialised to live on a diet of lignocellulosic biomass. These studies can help us to understand important environmental components of the global carbon cycle, as well as providing us with new understanding and tools to improve the industrial exploitation of lignocellulosic biomass.

The organisms we study include two species of crustacean woodborers (Limnoria tripunctata, and Chelura terebrans), a bivalve molluscan shipworm (Lyrodus pedicellatus) and an insect detritivore (Thermobia domestica). We used transcriptomic and proteomic analyses to study the genes and proteins expressed in the digestive system of these animals in order to identify the major digestive enzymes that they use. Our studies reveal clear similarities and novel features of the digestive proteome of the different species. Our work has already revealed a number of novel enzyme classes from invertebrate lignocellulose degraders. These include: the first report of processive cellobiohydrolases from animals, a new class of lytic polysaccharide monooxygenases (and the first reported in animals), and a new class of lignin active enzymes that speed up the digestion of wood by cellulases. Our studies show that the animals focus on digesting the cellulosic component of lignocellulose as this is made of polymers of glucose, which is easy to metabolise. However, in order to access the cellulose they have to disrupt the lignin and hemicellulose components that encase the cellulose. We have recently used 2-dimensional NMR studies of faecal residues from Limnoria and Lyrodus to identify major changes in wood composition during digestion. These reveal that there is extensive removal of the glucuronic acid and acetyl side chains of hemicellulose during digestion. Recent work from other groups suggests that the glucuronic acid side chains of xylans (a major hemicellulose) provide a link to the lignin, and that the acetyl esters help stabilise the associations between xylans and cellulose.

Our proposed work is to identify and characterise some of the, as-yet uncharacterised, major activities from our invertebrate systems. These include two families of glycosyls hydrolases (GHs) found in all four digestive systems (GH9s and GH30s) and which we have now been able to produce in a recombinant form, the lignin-modifying enzymes of Lyrodus (now available in recombinant forms), and acetyl esterases and glucuronidases from Lyrodus and Limnoria. We will use high-throughput robotic facilities in our lab to test combinations of enzymes from our collection for their ability to synergise the deconstruction of lignocellulosic and cellulosic substrates. We will also assess the ability of our enzymes to enhance the activity of commercial cellulase preparations and work with partners from the enzyme industry to develop ways to exploit our discoveries.

Technical Abstract:
Lignocellulosic biomass is the most abundant biological material on the planet due to its important role in providing support to land plants and its general recalcitrance to digestion. The turnover of lignocellulose in the environment is an important part of the global carbon cycle. In addition, lignocellulose from waste, such as agricultural crop residues, and paper and card that constitute a major biological components of municipal solid waste, represent a potential low carbon replacement for petroleum in the production of liquid fuels and chemicals. We have been studying the digestive systems of four different lignocellulose-consuming invertebrate species in order to understand how they derive nutrition from this recalcitrant material. These studies improve our understanding of the degradation of lignocellulose in nature, and also provide new enzymes and understanding of this process that can be applied in an industrial context for biorefining lignocellulose. Our work has led to significant discoveries of new enzymes for cellulose degradation and lignin modification, and revealed a number of common features in the digestive systems of wood degrading invertebrates, as well as distinct specialisations. Recent advances in recombinant protein production have opened the opportunity to explore new enzyme targets that we could not previously express. In addition, the application of two-dimensional nuclear magnetic resonance studies to the faecal products from our target invertebrates have revealed the importance of hemicellulose modifications to the digestive process (notably the removal of glucuronosyl and acetyl side chains from xylans). We now propose to bring our studies to fruition by expressing and characterising some of the remaining major enzymes from our invertebrate digestive systems. We will use of our high-throughput assay systems to examine the synergies between enzymes, and assemble effective lignocellulolytic cocktails.

Potential Impact:
The proposed work fits well with the BBSRC's strategic priorities in Industrial Biotechnology and Bioenergy. The provision of a source of sugars that can form the platform for fermentative production of biofuels and platform chemicals without having negative consequences for food security is a major challenge of our times. The work in this project is explicitly aimed at identifying new enzymes and processes for the saccharification of non-food lignocellulosic biomass.

The work described in this proposal will most immediately benefit the private commercial sector by identifying and developing new enzymes for use in industrial biotechnological applications. The primary beneficiaries will be those in the enzyme industry, however, if this work significantly improves lignocellulose saccharification, its impacts will trickle down to many areas of the developing knowledge-based bio-economy by providing cost-competitive lignocellulosic sugars for multiple applications. In addition, cellulases and related carbohydrate active enzymes are used very widely in other industrial sectors including, pulp and paper, textiles, laundry detergents and the food industry, and our work may have impacts in any or all of these.

Our work will benefit other researchers working in the areas of enzymes, polysaccharides, biofuels and industrial biotechnology and enzymes structure and function, by providing novel data on new examples of lignocellulose active enzymes from animals and particularly from the marine environment, a largely underexplored area.

Our work could have long-term benefits for the environment and society at large by helping to decrease our reliance on fossil resources and help provide lower carbon routes for the production of fuels and industrial chemical with minimal impacts on food security. This work therefore has potentially important impact from the political perspective, potentially helping the UK to reach its targets for reduced carbon emissions.
We have assembled an extremely strong group of scientists to maximise the chances of the full promise of the scientific research being realised. In addition, we are committed to seeing that the impacts of the work are maximised. This will, in part, be achieved by working closely with our industrial partner to ensure that enzymes have a route for rapid industrial uptake. In addition, the team has a good record in terms of public outreach and communication. The research on marine woodborers provides an excellent means of catching public interest as witnessed by numerous popular press articles that have been published on our previous work in this area.

We will use proven processes to protect IP and publish results in scientific journals and at conferences. We will also use existing UK networks (NIBB, KTNs etc.) to communicate progress through their events and web-based or printed media. When appropriate, discoveries will be disseminated by the University to the general media through press releases. To ensure professional management of intellectual property, CNAP operates regular IP reviews of all projects. CNAP has an outstanding track record in commercialisation of strategic research through on-going collaborations with companies throughout the biorenewable supply chain.
The programme will provide researchers with wide-ranging skills relevant to the establishment of a vibrant industrial biotechnology and bioenergy research and innovation-led industrial sector in the UK.