The development of sustainable biofuels based on woody (lignocellulosic) plant biomass is generally seen as an essential element in the move to a low carbon economy within the constraints of food security. The key to unlock this potential lies in developing technologies for the cost effective deconstruction of lignocellulose into sugars for fermentation. A number of animals have evolved to live on the large amounts of woody biomass that enter the marine environment from river estuaries. Such animals may provide useful understanding and enzymes for lignocellulose deconstruction, as they thrive on a diet of wood, and digest it under ambient temperatures and pressures.

The project builds on an existing programme funded as part of the BBSRC Sustainable Bioenergy Centre that has focused on Limnoria quadripunctata, a wood borer, which is unusual in having a digestive tract free of microbial life. Our studies indicate that this animal has a gut with an inert chitinous lining in which free radical chemistry is deployed as a pretreatment for enzymatic hydrolysis, and in which enzymes are produced in a separate organ and transposed to the gut, which functions as an enzyme reactor. In the programme we have established a wide range of specialist capabilities in the area of producing and characterising recombinant secreted Eukaryotic enzymes, as well as methods for studying digestive processes in vivo.

Building from this basis of knowledge, skills and resources, we wish to expand the work to take in a wider range of wood-eating marine invertebrates. Our initial understanding was that the microbe-free gut was peculiar to the Limnoriids, but recent studies at Portsmouth show a similar situation in a marine amphipod, Chelura telebrans. We have now obtained substantial EST databases from the digestive tracts of three arthropods, two Limnoriids (L. quadripunctata and L. lignorum), and C. telebrans. Initial scrutiny reveals some broad similarities in the suite of obvious digestive genes expressed in the three, but also considerable divergence in the range and relative expression levels of these genes. We will undertake detailed comparative gut and hepatopancreas transcriptomic and proteomic studies to identify the range of proteins involved in digestion and also study the biochemical processes in the gut. C. terebrans has the advantage of being considerably larger than the Limnoriid species, which will make these studies easier to undertake.

In addition to crustacean wood borers, we also propose to initiate studies in Lyrodus pedicellatus, a representative of the shipworms. Shipworms are large bivalve molluscs with little relationship to the arthropods. The bivalve shells of shipworms have evolved into a highly effective rasp at the anterior of the animal, with which they bore through wood, consuming particles as they go. These animals have a complex and poorly understood digestive that includes a large chamber where wood particles are stored and a region where wood particles are phagocytosed indicating intracellular digestion. In addition, shipworms harbour endo-symbiotic bacteria in their gills that have been suggested to aid in digestion, and have intestinal microflora that could also be involved. We will investigate genes and enzymes expressed, and biochemical processes in different regions of the shipworm digestive system in order to identify new enzymes for study. We will produce recombinant forms of hydrolytic and oxidative enzymes involved in wood digestion and characterise these in detail in terms of structure and activity. We will work closely with industry to examine the commercial potential of new enzymes and pretreatment approaches developed in the project.

Technical Abstract:
Our previous work has studied a marine wood boring crustacean (Limnoria quadripunctata) that has a digestive system that is free of microbial life This work has shown that L. quadripunctata has a two compartment digestive system, where enzymes for digestion are produced in the hepatopancreas, while wood digestion occurs in the hindgut, which is protected by the food mass being encased in a non-living lining. The hindgut environment has high levels of reactive oxygen species and is an oxygen sink, suggesting that oxidative chemistry is being used to attack the lignocellulose, and this may also be responsible for the lack of microbes. We have been characterising enzymes from the digestive system and studied a new animal GH7 cellulase in considerable detail. This enzyme shows remarkable robustness with higher activity in 3M NaCl than at lower concentrations. The enzyme has an unusually high density of acidic residues at its surface, and this feature is seen in other enzymes from this system and may be associated with robustness necessary in the inhospitable gut conditions.

We will build on our previous work and the resources established during it. We will extend our studies to two other crustacean wood borers, L. lignorum and Chelura terebrans, which we find to have microbe free digestive systems. We will also include a shipworm, a wood boring mollusc, that appears to use very different approach to wood digestion but has been little studied. We will use transcriptomic, proteomic, biochemical, microscopic and spectroscopic studies of the digestive systems in these organisms to provide understanding of the processes and identify interesting enzymes for deeper study. We will produce recombinant version of lignocellulose active enzymes that will be studied in structural and biochemical detail. We will work with our industrial partner to investigate the commercial potential of these enzymes, and test them at scale using pilot scale biorefinery facilities.

Potential Impact:
This programme fits well with the BBSRCs 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 helpingthe 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 records in terms of public outreach and communication. For example SMM has appeared in two recent Research Council videos and is a member of the BBSRC Bioenergy Outreach Group. The research on marine wood borers provides an excellent means of catching public interest as witnessed by numerous popular press articles that have been published on the BSBEC Marine Wood Borer Programme.

The project management will use proven processes to protect IP and publish results in scientific journals and at conferences. We will also use existing UK networks (eg the Bioscience KTN, the NNFCC) 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 and PhD students with wide-ranging skills relevant to the establishment of a vibrant industrial biotechnology and bioenergy research and innovation-led industrial sector in the UK. We will encourage the researchers and students to attend networking meetings organised by BSBEC and other relevant networks that may be established through the new BBSRC new initiatives in industrial biotechnology and bioenergy.