Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.gribble | carbohydrate degrading enzymes

The digestion of lignocellulose is attracting attention both in terms of basic research into its metabolism by microorganisms and animals, and also as a means of converting plant biomass into biofuels. Limnoriid wood borers are unusual because, unlike other wood-feeding animals, they do not rely on symbiotic microbes to help digest lignocellulose. The absence of microbes in the digestive tract suggests that limnoriid wood borers produce all the enzymes necessary for lignocellulose digestion themselves. In this study we report that analysis of ESTs from the digestive system of Limnoria quadripunctata reveals a transcriptome dominated by glycosyl hydrolase genes. Indeed, >20% of all ESTs represent genes encoding putative cellulases, including glycosyl hydrolase family 7 (GH7) cellobiohydrolases. These have not previously been reported in animal genomes, but are key digestive enzymes produced by wood-degrading fungi and symbiotic protists in termite guts. We propose that limnoriid GH7 genes are important for the efficient digestion of lignocellulose in the absence of gut microbes. Hemocyanin transcripts were highly abundant in the hepatopancreas transcriptome. Based on recent studies indicating that these proteins may function as phenoloxidases in isopods, we discuss a possible role for hemocyanins in lignin decomposition. biofuels | cellulase | hemocyanin | phenoloxidase | wood degradation

BackgroundBivalve teredinids inflict great destruction to wooden maritime structures. Yet no comprehensive study was ever carried out on these organisms in European coastal waters. Thus, the aims of this study were to: investigate the diversity of teredinids in European coastal waters; map their past and recent distributions to detect range expansion or contraction; determine salinity-temperature (S-T) requirements of species; flag, for future monitoring, the species that pose the greatest hazard for wooden structures.ResultsA total of nine teredinid species were found established in European coastal waters. Seven were considered cryptogenic, of unknown origin, and two were considered alien species. Teredo navalis and Nototeredo norvagica were the species with the widest distribution in European waters. Recently, T. navalis has been reported occurring further east in the Baltic Sea but it was not found at a number of sites on the Atlantic coast of southern Europe. The Atlantic lineage of Lyrodus pedicellatus was the dominant teredinid in the southern Atlantic coast of Europe. In the Mediterranean six teredinid species occurred in sympatry, whereas only three of these occurred in the Black Sea. The species that pose the greatest hazard to wooden maritime structures in European coastal areas are T. navalis and the two lineages of L. pedicellatus.ConclusionsCombined data from field surveys and from the literature made it possible to determine the diversity of established teredinid species and their past and recent distribution in Europe. The environmental requirements of species, determined using climatic envelopes, produced valuable information that assisted on the explanation of species distribution. In addition, the observed trends of species range extension or contraction in Teredo navalis and in the two lineages of Lyrodus pedicellatus seem to emphasise the importance of temperature and salinity as determinants of the distribution of teredinids, whereas their life history strategy seems to play an important role on competition.Teredo navalis and pedicellatus-like Lyrodus species should be monitored due to their destructive capability. The two alien species may expand further their distribution range in Europe, becoming invasive, and should also be monitored.

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