New tools for the realization of cost-effective liquid biofuels from plant biomass (revised costs)

从木质纤维生物质生产液体生物燃料提供了以环境友好的方式提供液体运输燃料的潜力，而不会损害粮食安全。木质纤维素主要由可转化为单糖的多糖组成，可通过微生物发酵生产乙醇和丁醇等酒精。从植物生物质中生产这种液体生物燃料目前受到将木质纤维素转化为可发酵糖(糖化)的成本的阻碍。显然需要新的更好的酶来糖化木质纤维。一些动物，如白蚁，可以靠木质纤维素生存，这表明它们已经克服了从这种顽固的底物中获得糖的问题。这些生物体一般依靠消化道中的细菌和原生动物来帮助消化木质纤维素。在海洋环境中的小型甲壳类木材钻孔虫Limnoriidae(也被称为Grible)中发现了这一规则的例外。这些动物可以以木质纤维素为食生存，并且具有有效无菌的消化道，这一点是不同寻常的。这表明，利姆诺氏菌不仅可以用自己的酶消化木质纤维素，而且消化道内的条件，与木质纤维素的消化相关，阻止微生物形成。利诺里亚木质纤维素消化的不同寻常的性质表明，在发现糖化的新见解和方法以及工业应用的新酶和新基因方面具有巨大的潜力。以此类推，白蚁的消化道可以被视为一个复杂的微生物反应器，用于消化木质纤维素，而利姆诺亚肠道是一个酶反应器，因此其本质上更接近于当前的工业系统。为了揭示木质纤维分解过程中表达的基因，我们对利姆诺里亚的消化道进行了深度转录测序。我们对超过28万个cDNA进行了测序，揭示了这一过程的惊人洞察力。利姆诺里亚肠道转录组主要由编码几种主要蛋白质的基因控制。编码糖基水解酶(将多糖转化为糖的酶)的基因占cDNA的近30%，推测的纤维素酶和纤维二糖水解酶(包括一些在动物基因组中从未见过的酶)占转录组的近25%。许多其他蛋白质类别的丰度都很高，这表明它们可能参与了消化过程。本文介绍的工作方案旨在确定利姆诺里亚木质纤维素消化的关键机制和成分，以便我们能够应用这一过程中的原理和酶来促进工业木质纤维糖化。为了更好地确定特定蛋白质在消化过程中的作用，我们将确定它们是否分泌到发生消化的肠腔中。我们将生产重组版本的酶，表征它们的酶特性，并确定它们对使用单个酶和组合进行木质纤维素糖化的有效性。我们还将确定，表达这些酶的基因是否可以用于提高能源作物中木质纤维生物质的糖化潜力。

The production of liquid biofuels from lignocellulosic biomass offers the potential to provide liquid transportation fuels in an environmentally benign manner without compromising food security. Lignocellulose is largely composed of polysaccharides that can be converted into simple sugars and used to produce alcohols such as ethanol and butanol by microbial fermentation. Production of such liquid biofuels from plant biomass is currently hampered by the cost of converting lignocellulose into fermentable sugars (saccharification). There is a clear need for new and better enzymes for lignocellulose saccharification. A number of animals such as termites can survive on a diet of lignocellulose, suggesting they have overcome the problem of obtaining sugars from this recalcitrant substrate. These organisms generally rely on a population of bacteria and protists in their digestive tract that help to digest the lignocellulose. An exception to this rule is found in the Limnoriidae (also known as gribble), small crustacean wood borers from the marine environment. These animals can survive on a diet of lignocellulose and are unusual in having an effectively sterile digestive tract. This suggests that not only can Limnoria digest lignocellulose with their own enzymes, but that conditions within the digestive tract, associated with lignocellulose digestion, prevent microbes from becoming established. The unusual nature of lignocellulose digestion in Limnoria indicates a great potential for uncovering new insights and approaches to saccharification and new enzymes and genes for industrial applications. By analogy, the termite digestive tract can be seen as a complex microbial reactor for lignocellulose digestion, whereas the Limnoria gut is an enzyme reactor, and thereby much closer in its nature to current industrial systems. We have used deep transcriptomic sequencing of the digestive tract of Limnoria in order to reveal the genes expressed during lignocellulose breakdown. We sequenced more than 280,000 cDNAs revealing a breathtaking insight into this process. The Limnoria gut transcriptome is dominated by genes encoding several major classes of protein. Genes encoding glycosyl hydrolases (enzymes that convert polysaccharides into sugars) account for almost 30% of cDNAs, and putative cellulases and cellobiohydrolases (including some never before seen before in animal genomes) account for almost 25% of the transcriptome. A number of other protein classes are represented at very high abundance suggesting they may be involved in the digestive process. The programme of work presented here aims to identify the key mechanisms and components of lignocellulose digestion in Limnoria, in order that we can apply principles and enzymes from this process in order to enhance industrial lignocellulose saccharification. To better determine the roles of particular proteins in the digestive process we will establish whether or not they are secreted into the gut lumen where digestion occurs. We will produce recombinant versions of the enzymes, characterise their enzymatic properties and determine their usefulness for lignocellulose saccharification both using individual enzymes as well as combinations. We will also establish whether expressing the genes encoding these enzymes can be used to improve the saccharification potential of lignocellulosic biomass in energy crops.