Harnessing invertebrate wood digestion systems

木质纤维生物质是生物圈内最大的固定碳储量，其降解是全球碳循环的一个重要组成部分。以作物残渣和城市固体废物的生物部分的形式存在的木质纤维素也是生产可持续燃料、化学品和材料的石油的潜在低碳替代品。木质纤维素主要由多糖和木质素组成，是糖类和酚类物质的丰富潜在来源。这些化合物可以作为以其为食的有机体的饮食营养来源，也可以作为工业的平台化学品，但在这两种情况下，由于木质纤维素的顽固性，获得这些化合物都是具有挑战性的。我们正在研究专门以木质纤维生物质为食物的无脊椎动物的消化系统。这些研究有助于我们了解全球碳循环的重要环境因素，并为我们提供新的认识和工具，以促进木质纤维生物的工业化开发。我们研究的生物包括两种甲壳类伐木害虫(Limnoria tripot tata和Cherura terebrans)、一种双壳类软体动物船虫(Lyrodus Pedicellatus)和一种有害昆虫(Thermobia Home Tica)。我们使用转录组学和蛋白质组学分析来研究这些动物消化系统中表达的基因和蛋白质，以确定它们使用的主要消化酶。我们的研究揭示了不同物种消化蛋白质组的明显相似之处和新的特征。我们的工作已经揭示了无脊椎动物木质纤维素降解物中的一些新的酶类。其中包括：首次报道动物纤维二糖水解酶，一类新的裂解多糖单加氧酶(也是第一次在动物中报道)，以及一类新的木质素活性酶，可加速纤维素酶对木材的消化。我们的研究表明，动物专注于消化木质纤维素的纤维素成分，因为这是由葡萄糖聚合物组成的，这种聚合物很容易代谢。然而，为了获得纤维素，它们必须破坏包裹纤维素的木质素和半纤维素成分。我们最近对利姆诺里亚和莱洛德斯的粪便残留物进行了二维核磁共振研究，以确定在消化过程中木材成分的主要变化。这表明，在消化过程中，半纤维素的葡萄糖糖醛酸和乙酰侧链被广泛去除。最近来自其他研究小组的工作表明，木聚糖(一种主要的半纤维素)的葡萄糖醛酸侧链提供了与木质素的联系，而乙酰酯有助于稳定木聚糖和纤维素之间的联系。我们建议的工作是识别和表征我们无脊椎动物系统中的一些尚未确定的主要活动。其中包括在所有四个消化系统(GH9S和GH30S)中发现的两个糖基水解酶(GHS)家族，我们现在已经能够以重组形式生产它们，Lyrodus的木质素修饰酶(现已获得重组形式)，以及来自Lyrodus和Limnoria的乙酰酯酶和葡萄糖醛酸酶。我们将使用实验室中的高通量机器人设施来测试我们收集的酶的组合，以了解它们协同分解木质纤维和纤维素底物的能力。我们还将评估我们的酶提高商业纤维素酶制剂活性的能力，并与酶行业的合作伙伴合作开发利用我们的发现的方法。

Lignocellulosic biomass represents the greatest reserve of fixed carbon in the biosphere and its degradation is an important component of the global carbon cycle. Lignocellulose, in the form of crop residues and the biological fraction of municipal solid waste, also represents a potential low-carbon replacement for petroleum for the production of sustainable fuels, chemicals and materials. Lignocellulose is a rich potential source of sugars and phenolics as it is primarily comprised of polysaccharides and lignin. These can serve as a source of dietary nutrition for organisms that feed on it, or as platform chemicals for industry, but in both cases, obtaining these compounds is challenging due to the recalcitrance of lignocellulose. We are studying the digestive systems of invertebrate animals specialised to live on a diet of lignocellulosic biomass. These studies can help us to understand important environmental components of the global carbon cycle, as well as providing us with new understanding and tools to improve the industrial exploitation of lignocellulosic biomass.The organisms we study include two species of crustacean woodborers (Limnoria tripunctata, and Chelura terebrans), a bivalve molluscan shipworm (Lyrodus pedicellatus) and an insect detritivore (Thermobia domestica). We used transcriptomic and proteomic analyses to study the genes and proteins expressed in the digestive system of these animals in order to identify the major digestive enzymes that they use. Our studies reveal clear similarities and novel features of the digestive proteome of the different species. Our work has already revealed a number of novel enzyme classes from invertebrate lignocellulose degraders. These include: the first report of processive cellobiohydrolases from animals, a new class of lytic polysaccharide monooxygenases (and the first reported in animals), and a new class of lignin active enzymes that speed up the digestion of wood by cellulases. Our studies show that the animals focus on digesting the cellulosic component of lignocellulose as this is made of polymers of glucose, which is easy to metabolise. However, in order to access the cellulose they have to disrupt the lignin and hemicellulose components that encase the cellulose. We have recently used 2-dimensional NMR studies of faecal residues from Limnoria and Lyrodus to identify major changes in wood composition during digestion. These reveal that there is extensive removal of the glucuronic acid and acetyl side chains of hemicellulose during digestion. Recent work from other groups suggests that the glucuronic acid side chains of xylans (a major hemicellulose) provide a link to the lignin, and that the acetyl esters help stabilise the associations between xylans and cellulose.Our proposed work is to identify and characterise some of the, as-yet uncharacterised, major activities from our invertebrate systems. These include two families of glycosyls hydrolases (GHs) found in all four digestive systems (GH9s and GH30s) and which we have now been able to produce in a recombinant form, the lignin-modifying enzymes of Lyrodus (now available in recombinant forms), and acetyl esterases and glucuronidases from Lyrodus and Limnoria. We will use high-throughput robotic facilities in our lab to test combinations of enzymes from our collection for their ability to synergise the deconstruction of lignocellulosic and cellulosic substrates. We will also assess the ability of our enzymes to enhance the activity of commercial cellulase preparations and work with partners from the enzyme industry to develop ways to exploit our discoveries.