Discovering Novel Lignocellulose Degrading Enzymes from the Marine Wood Borer, Limnoria quadripunctata

Transportation accounts for a large proportion of global CO2 emissions, estimated at 22% of all anthropogenically produced CO2 in 2010, with most of this coming from the use of liquid transportation fuels. Second generation biofuels offer a sustainable opportunity to decarbonise this sector of global energy use. To realise the potential of second generation biofuels more efficient methods of deconstructing their lignocellulosic starting material must be developed. Understanding how the marine wood borer, Limnoria quadripunctata, is able to do this biochemically without the assistance of microbial symbionts may inspire new techniques to achieve this in an industrial setting. Whilst the anatomy of Limnoria has been well studied, and the transcriptome of its hepatopancreas (HP), a secretory organ of the digestive system, has been previously elucidated, little is known about how the animal is able to derive nutrients from its diet of wood without the aid of microbial symbionts. In this work a proteomic study of the HP and gut tissue of Limnoria was carried out and the data generated analysed in concert with that of the HP transcriptome to identify proteins which may be involved in lignocellulose digestion in the animal. In this way the glycosyl hydrolases, hemocyanins (HCs), ferritins and leucine rich repeat proteins were identified as having the potential to be involved in lignocellulose digestion. Work was undertaken to characterise the function of HC proteins in Limnoria. Reverse transcriptase quantitative polymerase chain reaction analysis showed all five Limnoria HC genes to be solely expressed in the HP, whilst Western blot and proteomic examination showed HC protein to be present in the HP and gut, as well as the rest of the body. This indicates that HCs are transported post translation to the gut, the site where ingested wood particles are segregated and presumably digested. In vivo experiments suggested that a phenoloxidase enzyme activity was associated with the production of peroxides in the gut of Limnoria. Using a soluble extract of Limnoria tissue a di-phenoloxidase (DPO) enzyme activity was demonstrated which it was possible to inhibit using a phenoloxidase inhibitor. Mass spectrometry analysis implicated HC protein as the species responsible for this activity. Multiple sequence alignment showed that the Limnoria HC sequences possess the conserved features associated with the ability to carry out a DPO enzyme activity described in HC proteins from other arthropod species. Attempts were made to heterologously express Limnoria HC proteins in a number of systems with limited success being achieved using the ArcticExpress strain of Escherichia coli. Partially pure heterologous protein produced in this way was able to show a DPO enzyme activity which was inhibited by a phenoloxidase inhibitor. Mass spectrometry analysis implicates HC as the protein species responsible for this activity. Based on the findings of this work it seems possible that the hemocyanin proteins of Limnoria aid the animal in digesting wood by contributing to the formation of reactive peroxide compounds in its gut which attack lignocellulose. This process may also contribute towards the sterility of the gut by creating an inhospitable environment for microbial colonisation. Further work is now required to determine the mechanisms by which it occurs.