In silico study of lignocellulosic biofuel processes

从理论上讲，基因组序列提供了定义感兴趣的生物系统结构所需的所有信息。例如，知道一个细胞中的所有酶和每个酶接受的底物以及每个酶可以产生的所有产品，就有可能形成一个代表该细胞内可能的生化反应系统的完整体系的生物反应主全球网络。在这项研究中，我们计划使用一个‘基因组规模’的代谢网络(GSMN)，这是从一些在生物燃料生产中应用的物种的序列数据中重建的。已经为一些医学和工业上重要的物种出版了GSMN，包括天蓝色链霉菌和结核分枝杆菌(我们的这2个)，分别促进了工艺设计和抗生素靶标的识别的新方法。我们计划使用基因组规模的建模来演示在电子实验中对生物精炼的效用。该方法将把能够进行木质纤维素降解的基因组与能够生产生物燃料的基因组联系起来，以期预测形成体内研究基础的过程。据我们所知，这将是第一个以这种方式连接基因组规模模型的项目，因此除了提供实用信息外，还将代表着一项科学进步。这两个阶段(生物质降解，然后是单独的生物乙醇生产阶段)可能比单一的微生物处理步骤更有效。生物量降解最近对两种“模式生物”(真菌里氏木霉和细菌热梭菌)的基因组进行了测序，因此，这些物种将被纳入这项研究。然而，考虑到目前木质纤维素降解对酸和热处理的依赖，在低pH值和高温下稳定和活性的酶具有特殊的价值。因此，从嗜热和嗜酸生物中提取的降解木质纤维素的酶在工业过程中具有重要的应用前景，因此，Caldicellulosiruptor charcharolyticus和Acidothermus carcharolyticus(两者都已被测序)将被包括在生物质降解阶段。生物燃料生产一个重要的产量限制因素是乙醇对发酵宿主的毒性。大多数发酵生物，如酿酒酵母，不能耐受高浓度的乙醇，导致产品必须通过昂贵且能源密集型的蒸馏步骤进行浓缩。树干毕赤酵母代表了一种与生物燃料研究相关的酵母物种，其基础是其发酵木糖的天然能力。它最近测序的基因组揭示了对这一过程负责的代谢途径的洞察，该物种将被包括在我们的第二阶段移动发酵单胞菌模型中，该生物单胞菌被描述为具有相当大的生物燃料生产潜力最近也被测序并将被包括在最后，使用大肠杆菌，它已经被设计为使用非发酵途径生产异丁醇和其他酒精，将被包括在内。将研究生物质降解的原料将包括木质纤维素(经过和不经过化学前处理)和共基质，这可能会提高代谢的生物能量效率。这四个物种中的每一个都将构建GSMN。在每种情况下，将准备(使用序列注释)GSMN‘草稿’，该‘草稿’将通过添加进一步的反应来精炼，以便‘关闭’模型。每个GSMN包括许多(通常是数百个)“输入门”，即潜在的底物，由基因序列预测，但从未在实验室尝试过。在硅胶模型中使用，我们将能够检查底物组合的效果，这将需要在体内进行多年的实验。

In theory, a genome sequence provides all of the information necessary to define the structure of the biological system of interest. For example, knowing all of the enzymes in a cell and the substrates that each one accepts and all of the products that each one can make, it is possible to formulate a bioreaction master global network that represents the complete repertoire of possible biochemical reaction systems within that cell. In this study, we plan to use a 'genome-scale' metabolic network (gsmn), reconstructed from the sequence data for a number of species with applications in biofuel production. Gsmns have already been published for a number of medically- and industrially-important species, including Streptomyces coelicolor and Mycobacterium tuberculosis (these 2 by us) facilitating novel approaches to process design and identification of antibiotic targets, respectively. We plan to use genome scale modelling to demonstrate the utility of in silico experimentation to Biorefining. The approach will link genomes, capable of carrying out lignocellulose degradation to genomes able to produce biofuels, with a view to predicting processes that will form the basis for an in vivo study. As far as we are aware, this will be the first project to link genome scale models in this way, and will therefore represent a scientific advance in addition to providing pragmatic information. This 2-stage (biomass degradation followed by a separate bioethanol production stage) is potentially more efficient than a single microbial processing step. Biomass Degradation The genomes of two 'model organisms' (the fungus Trichoderma reesei and the bacterium Clostridium thermocellum) have recently been sequenced and these species will, therefore, be included in the study. However, considering the current dependence on acid and heat pre-treatment in lignocellulose degradation, enzymes that are stable and active at low pH values and at high temperatures are of particular value. Thus, enzymes derived from thermophilic and acidophilic organisms known to degrade lignocellulose hold significant promise for industrial processes, and, for this reason Caldicellulosiruptor saccharolyticus and Acidothermus cellulolyticus (both of which have been sequenced) will be included in the biomass degradation stage. Biofuel production A significant yield limiting factor is the toxicity of ethanol to the fermenting host. Most fermenting organisms such as S. cerevisiae cannot tolerate high ethanol concentrations resulting in a product that must then be concentrated through an expensive and energy-intensive distillation step. Pichia stipitis represents one yeast species of relevance to biofuel research based on its natural ability to ferment xylose. Its recently sequenced genome revealed insights into the metabolic pathways responsible for this process and this species will be included in our second stage modelling Zymomonas mobilis, which has been described as having considerable potential for biofuel production has also been sequenced recently and will be included Finally, the use of E. coli, which has been engineered to produce isobutanol and other alcohols, using non-fermentative pathways, will be included. The feed-stocks to be examined for biomass degradation will include lignocellulose (with and without chemical pre-treatment) and co-substrates, which may enhance bioenergetic efficiency of metabolism. Each of the four species will have gsmns constructed. In each case, a 'draft' gsmn will be prepared (using the sequence annotations) which will be refined by the addition of further reactions in order to 'close' the model. Each gsmn includes numerous (often hundreds of) 'input gates', ie potential substrates, predicted by the gene sequence but never tried in the laboratory. Using in silico models, we will be able to examine the effect of combinations of substrates that would require many years of experimentation in vivo.