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

从木质纤维素生物质中生产液体生物燃料，有可能在不损害粮食安全的情况下，以无害环境的方式提供液体运输燃料。木质纤维素主要由多糖组成，这些多糖可以转化为单糖，并通过微生物发酵用于生产乙醇和丁醇等醇。从植物生物量生产这种液体生物燃料目前受到木质纤维素转化为可发酵糖（糖化）成本的阻碍。显然，需要新的、更好的酶来进行木质纤维素的糖化。许多动物，如白蚁，可以以木质纤维素为食而生存，这表明它们已经克服了从这种顽固的底物中获取糖的问题。这些生物通常依靠消化道中的细菌和原生生物来帮助消化木质纤维素。这条规则的一个例外是Limnoriidae（也被称为gribble），一种来自海洋环境的小型甲壳类木蛀虫。这些动物可以以木质纤维素为食而生存，并且具有有效无菌的消化道。这表明，Limnoria不仅可以用自己的酶消化木质纤维素，而且消化道内与木质纤维素消化相关的条件也阻止了微生物的形成。Limnoria中木质纤维素消化的不寻常性质表明，在揭示糖化的新见解和新方法以及用于工业应用的新酶和基因方面具有巨大的潜力。通过类比，白蚁的消化道可以被看作是木质纤维素消化的复杂微生物反应器，而沼虾的肠道是一个酶反应器，因此在其性质上更接近于目前的工业系统。为了揭示木质纤维素分解过程中表达的基因，我们对Limnoria的消化道进行了深度转录组测序。我们对超过280,000个cdna进行了测序，揭示了对这一过程的惊人见解。Limnoria肠道转录组由编码几种主要蛋白质的基因主导。编码糖基水解酶（将多糖转化为糖的酶）的基因约占cdna的30%，而推定的纤维素酶和纤维素生物水解酶（包括一些从未在动物基因组中见过的酶）约占转录组的25%。许多其他种类的蛋白质都有很高的丰度，这表明它们可能与消化过程有关。本文提出的工作计划旨在确定Limnoria中木质纤维素消化的关键机制和组成部分，以便我们可以应用该过程中的原理和酶来提高工业木质纤维素糖化。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消化发生的肠腔中。我们将生产这些酶的重组版本，表征它们的酶特性，并确定它们在使用单个酶和组合酶进行木质纤维素糖化的有用性。我们还将确定表达编码这些酶的基因是否可以用于提高能源作物中木质纤维素生物质的糖化潜力。

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.