Relating fermentation pathways to energy conservation in lignocellulolytic clostridia and related organisms

以石油为基础的液体运输燃料的替代品是植物乙醇。从植物生物质(秸秆、木材等)中提取的木质纤维素是生产乙醇所需的可发酵糖的丰富来源，尽管这是一种顽固的来源。目前的方法包括生产酵母菌发酵糖的复杂前处理。一种替代的强化生物工艺可以使用发酵纤维素的热梭状芽孢杆菌直接从木质纤维素中生产乙醇。代谢模型一直是一个问题，因为它的“非典型”糖酵解途径，使用GTP和焦磷酸以及三磷酸腺苷，缺乏丙酮酸激酶。它也是一种严格的厌氧菌，可以降解半纤维素，但不能发酵。如果与发酵木聚糖的杆菌和耐空气的纤维素降解菌株合作，这些问题可能会得到解决。然而，在生物化学方面，后一类生物的特性很差。*这个项目的目标是了解这3个相关生物体的能量守恒途径。为实现目标，该项目将培养2名博士、2名硕士。和5名本科生HQP。*目标是：*A)确定中枢代谢途径的酶的作用，特别是关于所使用的能量辅助因子(GTP、ATP、焦磷酸盐)，以便更好地了解它们独特的代谢作用。*B)了解这些生物在以木质纤维素为碳源的共培养中的相互作用。*我们建议完成在前一次Discovey拨款中开始的、与糖酵解相关的能量守恒相关酶的表征。生长条件特异性表达--与“非典型”糖酵解相关的关键基因产物的差异将通过RTqPCR方法以及不同能量分子的相互作用来确定。这样的数据将导致纤维素发酵的强有力的代谢模型。一种类似的方法将被用于确定相似之处并突出不同之处。当这些生物作为混合生物膜在固体纤维底物上相互作用时，表达模式的变化将使用蛋白质组学方法进行研究。它们的空间分布将用荧光原位杂交方法观察。一种假说认为，由于表面纤维素酶的不同构型，斯特氏梭菌可能是中上层的，而携带纤维素体的高温梭菌和斯特氏梭菌可能是附着的。这些实验还将使用甲烷热自养杆菌作为与纤维素降解环境相关的终端氢受体，以及衰弱Caldibacillusdebilis，这是一种兼性好氧细菌，也与环境中的高温梭菌有关。通过这项工作产生的对代谢的理解将导致开发生物和发酵策略，有望提高从纤维素生物质生产乙醇的效率，以促进绿色经济。

A substitute for petroleum-based liquid transportation fuel is plant-based ethanol. An abundant, albeit recalcitrant, source of fermentable sugar for ethanol production is lignocellulose derived from plant biomass (straw, wood, etc.). Present methods involve complex pre-treatments to produce yeast-fermentable sugar. An alternate consolidated bioprocess can use cellulose-fermenting Clostridium thermocellum to produce ethanol directly from lignocellulose. Metabolic modelling has been an issue because of its “atypical” glycolytic pathway, which uses GTP and pyrophosphate along with ATP, and lacks pyruvate kinase. It is also a strict anaerobe that can hydrolyse but not ferment hemi-cellulose. These issues may be resolved if partnering with the xylan-fermenting C. stercorarium and the air-tolerant cellulose degrading C. straminisolvens. Biochemically, however, these latter organisms are poorly characterized. ***The goal of this program is to understand the energy conservation pathways in these 3 related organisms. In achieving its goal, this program will train 2 Ph.D., 2 M.Sc. and 5 undergraduate HQP.***The objectives are: ***A) To define the roles of the enzymes of pathways of central metabolism, especially with respect to the energy cofactors used (GTP, ATP, pyrophosphate) in order to better understand their distinctive metabolic roles.***B) To understand interactions of these organisms when grown in co-cultures on lignocellulose as the carbon source. ***We propose to complete the characterization of energy conservation associated enzymes of glycolysis in C. thermocellum started in the previous Discovey grant. Growth-condition-specific expression-differences of key gene-products associated with "atypic" glycolysis will be determined by RTqPCR methods, as well as interplay of the different energy molecules. Such data will lead to robust metabolic models for cellulose fermentation. A similar approach will the used for C. stercorarium and C. straminivolvens to confirm similarities and highlight differences. Expression pattern changes when these organisms interact as mixed biofilms on solid cellulosic substrates will be studied using proteomic approaches. Their spatial distribution will be observed using fluorescent-in situ-hybridization methods. One hypothesis is that C. stercorarium may be pelagic because of the different architecture of their surface cellulases, while C. thermocellum and C. straminisolvens, which both carry cellulosomes may be attached. These experiments will also be performed using Methanothermobacter thermoautotrophicus as a terminal hydrogen acceptor associated with cellulose degrading environments, as well as Caldibacillus debilis, a facultative aerobe also associated with C. thermocellum in the environmental. The metabolic understanding generated through this work will lead to developing organisms and fermentation strategies that are expected to improve the efficiency of ethanol production from cellulosic biomass for the Green Economy.*****