Learning from marine wood borers; enzymes and mechanisms of lignocellulose digestion

以木本(木质纤维素)植物生物质为基础的可持续生物燃料的开发通常被视为在粮食安全的限制下转向低碳经济的基本要素。释放这一潜力的关键在于开发技术，以经济高效的方式将木质纤维素分解为糖类进行发酵。许多动物已经进化到依靠从河口进入海洋环境的大量木质生物质为生。这些动物可能会为分解木质纤维素提供有用的理解和酶，因为它们以木材为食，并在环境温度和压力下消化木质纤维。该项目建立在现有项目的基础上，该项目是BBSRC可持续生物能源中心的一部分，该中心专注于四点利姆诺亚，一种木材钻孔虫，它的消化道不存在微生物生命，这是不寻常的。我们的研究表明，这种动物的肠道具有惰性几丁质内层，其中自由基化学被用作酶水解的预处理，其中的酶在单独的器官中产生并转位到肠道，后者起到酶反应的作用。在该计划中，我们在生产和鉴定重组分泌型真核生物酶方面建立了广泛的专业能力，并建立了研究活体消化过程的方法。在此基础上，我们希望扩大工作范围，以涵盖更广泛的以木材为食的海洋无脊椎动物。我们最初的理解是，这种无微生物的肠道是莱姆诺利虫特有的，但朴茨茅斯最近的研究表明，在海洋端足类动物切鲁拉长臂猿中也有类似的情况。我们现在已经从三种节肢动物、两种粘虫(四点粘虫和木头粘虫)和长尾轮虫的消化道中获得了大量的EST数据库。初步研究发现，这三个基因表达的一系列明显的消化基因有一些广泛的相似性，但在这些基因的范围和相对表达水平上也有相当大的差异。我们将进行详细的肠道和肝胰腺转录和蛋白质组学比较研究，以确定参与消化的蛋白质范围，并研究肠道中的生化过程。Terebrans的优势是比Limnoriid物种大得多，这将使这些研究更容易进行。除了甲壳类木材钻孔虫外，我们还建议启动对Lyrodus pedicellatus的研究，Lyrodus pedicellatus是这种船虫的代表。船虫是大型双壳软体动物，与节肢动物关系不大。船虫的双壳类贝壳在动物的前部进化成了一种高效的刺刀，它们用它穿透木头，在前进的过程中消耗颗粒。这些动物有一个复杂而鲜为人知的消化系统，包括一个储存木屑的大房间和一个吞噬木屑的区域，表明细胞内的消化。此外，船虫的鳃中含有被认为有助于消化的内生共生菌，肠道微生物群也可能参与其中。我们将研究船虫消化系统不同区域表达的基因和酶，以及生化过程，以确定新的酶用于研究。我们将生产与木材消化有关的重组形式的水解酶和氧化酶，并从结构和活性方面对其进行详细的表征。我们将与工业界密切合作，研究项目中开发的新酶和前处理方法的商业潜力。

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.