New tools for the realization of cost-effective liquid biofuels from plant biomass (revised costs)

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. We have used deep transcriptomic sequencing of the digestive tract of Limnoria in order to reveal the genes expressed during lignocellulose breakdown. We sequenced more than 280,000 cDNAs revealing a breathtaking insight into this process. The Limnoria gut transcriptome is dominated by genes encoding several major classes of protein. Genes encoding glycosyl hydrolases (enzymes that convert polysaccharides into sugars) account for almost 30% of cDNAs, and putative cellulases and cellobiohydrolases (including some never before seen before in animal genomes) account for almost 25% of the transcriptome. A number of other protein classes are represented at very high abundance suggesting they may be involved in the digestive process. The programme of work presented here 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. To better determine the roles of particular proteins in the digestive process we will establish whether or not they are secreted into the gut lumen where digestion occurs. We will produce recombinant versions of the enzymes, characterise their enzymatic properties and determine their usefulness for lignocellulose saccharification both using individual enzymes as well as combinations. We will also establish whether expressing the genes encoding these enzymes can be used to improve the saccharification potential of lignocellulosic biomass in energy crops.

The cost effective saccharification of lignocellulosic biomass is recognized as a pivotal technical hurdle that must be overcome before the production of liquid biofuels from lignocellulosic biomass becomes a commercial reality. The search for new enzymes to disrupt lignin and release sugars from lignocellulose is being pursued aggressively around the world. We have identified a unique opportunity for the UK to become a major contributor to this competitive field. Limnoria are crustacean wood borers that can digest lignocellulose, and are unusual in having an effectively sterile digestive tract. This indicates that they can digest lignocellulose without the aid of resident gut flora in contrast to animals such as termites, which depend on such microbes. We have established the potential for gene and enzyme discovery in this organism by the provision of a potentially comprehensive transcriptomic database comprising over 280,000 ESTs. Initial analysis of the data reveals a transcriptome dominated by abundant transcripts representing several broad classes of proteins. Glycosyl hydrolases from 12 different families account for more than 25% of the ESTs, with two classes of putative cellulases and cellobiohydrolases alone accounting for more than 20%. Sequences with high homology to hemocyanins and phenol oxidases account for a further 17% of the transcriptome, followed by sequences with homology to proteases, ferritin, oxygenases, and a group with low homology to fatty acid binding proteins. We will study the digestive process in Limnoria in detail using proteomic and metabolomic analyses, and will characterize the enzymatic activities of recombinant proteins representing many of the genes involved in the digestive process. These enzymes will be assessed for utility in industrial lignocellulose saccharification, and we will investigate the use of these genes expressed in transgenic plants to improve the saccharification potential of lignocellulosic feedstocks.