The production of liquid biofuels from lignocellulosic biomass offers the potential to provide liquid transportation fuels in an environmentally benign manner without compromising food security. Lignocellulose is largely composed of polysaccharides that can be converted into simple sugars and used to produce alcohols such as ethanol and butanol by microbial fermentation. Production of such liquid biofuels from plant biomass is currently hampered by the cost of converting lignocellulose into fermentable sugars (saccharification). There is a clear need for new and better enzymes for lignocellulose saccharification. A number of animals such as termites can survive on a diet of lignocellulose, suggesting they have overcome the problem of obtaining sugars from this recalcitrant substrate. These organisms generally rely on a population of bacteria and protists in their digestive tract that help to digest the lignocellulose. An exception to this rule is found in the Limnoriidae (also known as gribble), small crustacean wood borers from the marine environment. These animals can survive on a diet of lignocellulose and are unusual in having an effectively sterile digestive tract. This suggests that not only can Limnoria digest lignocellulose with their own enzymes, but that conditions within the digestive tract, associated with lignocellulose digestion, prevent microbes from becoming established. The unusual nature of lignocellulose digestion in Limnoria indicates a great potential for uncovering new insights and approaches to saccharification and new enzymes and genes for industrial applications. By analogy, the termite digestive tract can be seen as a complex microbial reactor for lignocellulose digestion, whereas the Limnoria gut is an enzyme reactor, and thereby much closer in its nature to current industrial systems. The programme of work aims to identify the key mechanisms and components of lignocellulose digestion in Limnoria, in order that we can apply principles and enzymes from this process in order to enhance industrial lignocellulose saccharification.
We have used transcriptomic and proteomic studies to identify the major groups of enzymes involved in the didestion of wood by Limnoria. This revealed that some 25% of the digestive transcriptome encode a range of putative glycosyl hydrolase enzymes including a class of cellulase not previously found in animals. These GH7 cellulases account for 12% of the digestive transcrptome and dominate the digestive proteome. We produced recombinant versions of one of these cellulases and showed it to be a processive cellobiohuydrolase with remarkable resistance to high salt concentrations. Studies of the digestive system show that the process of wood digestion is potentiated by oxidative free radical attack in the cuticle-lined hindgut and this process probably accounts for the lack of microbial life in the hindgut. we have shown that hemocyanins (normally associated with oxygen transport) are found in the hindgut and capable of producing hydrogen peroxide, which can initiate highly reactive Fenton Chemistry in the presence of transition metals such as iron. We showed that the lignin in wood is radicalised during digestion indicating that Fenton chemistry ioccurrs in the hindgut. Free radical chemistry aids in the accewss of enzymes to the polysaccharides in the wood and we have shown that the animal's cellulases mobilise almost 50% of the cellulose in the wood during digestion. These highly novel findings have led to high impact publications and also underpinned a successful application for an sLOLA project to follow up on these findings. The success of this work has enabled us to obtain additional funding from the BBSRC and EC to support our studies on lignocellulose digestion and biorefining.