Harnessing invertebrate wood digestion systems

木质纤维素生物质是生物圈中最大的固定碳储量，其降解是全球碳循环的重要组成部分。以作物残余物和城市固体废物的生物部分形式存在的木质纤维素也是石油的潜在低碳替代品，可用于生产可持续燃料、化学品和材料。木质纤维素是糖和酚类物质的丰富潜在来源，因为它主要由多糖和木质素组成。这些可以作为以其为食的生物体的膳食营养来源，或作为工业的平台化学品，但在这两种情况下，由于木质纤维素的难降解性，获得这些化合物是具有挑战性的。我们正在研究专门以木质纤维素生物质为食的无脊椎动物的消化系统。这些研究可以帮助我们了解全球碳循环的重要环境组成部分，以及为我们提供了新的认识和工具，以提高工业开发的木质纤维素生物质。我们研究的生物体包括两种甲壳类木材蛀虫（Limnoria tripunctata和Chelura terebrans），一种双壳软体动物船蛆（Lyrodus pedicellatus）和一种昆虫食腐动物（Thermobia arctica）。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性和新特征。我们的工作已经揭示了一些新的酶类从无脊椎动物木质纤维素降解。其中包括：首次报道了来自动物的进行性纤维二糖水解酶、一类新的溶解性多糖单加氧酶（并且首次在动物中报道）和一类新的木质素活性酶，其加速了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，易于代谢。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组分。我们最近使用了二维NMR研究的粪便残留物从Limnoria和Lyrodus，以确定木材组成的主要变化在消化过程中。这些表明，在消化过程中，半纤维素的葡萄糖醛酸和乙酰基侧链被大量去除。最近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一个链接到木质素，和乙酰酯有助于稳定木聚糖和cellulose.We之间的协会提出的工作是确定和验证一些，尚未表征，从我们的无脊椎动物系统的主要活动。这些包括在所有四个消化系统中发现的两个糖基水解酶（GH）家族（GH 9和GH 30），并且我们现在已经能够以重组形式生产，Lyrodus的木质素修饰酶（现在可以重组形式获得），以及来自Lyrodus和Limnoria的乙酰酯酶和葡萄糖醛酸苷酶。我们将在实验室中使用高通量机器人设施来测试我们收集的酶的组合，以确定它们协同解构木质纤维素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.