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

石油基液体运输燃料的替代品是植物基乙醇。用于乙醇生产的可发酵糖的丰富（尽管是柠檬酸）来源是源自植物生物质（稻草、木材等）的木质纤维素。目前的方法涉及复杂的预处理以生产酵母可发酵糖。一种替代的联合生物工艺可以使用纤维素发酵热纤梭菌直接从木质纤维素生产乙醇。代谢建模一直是一个问题，因为它的“非典型”糖酵解途径，其中使用GTP和焦磷酸沿着ATP，缺乏丙酮酸激酶。它也是一种严格的厌氧菌，可以水解但不能发酵半纤维素。如果与木聚糖发酵菌C. stercorarium和耐气纤维素降解菌C. straminsolvens。然而，在生物化学上，这些生物体的特征很差。* 该计划的目标是了解这3种相关生物体的能量守恒途径。在实现其目标，该计划将培养2博士，2名理学硕士和5名HQP本科生 *目标是：*A）确定中枢代谢途径中酶的作用，特别是关于所使用的能量辅因子（GTP，ATP，焦磷酸），以便更好地理解其独特的代谢作用。B）了解这些生物在以木质纤维素作为碳源的共培养物中生长时的相互作用。* 我们建议完成C中糖酵解的能量守恒相关酶的表征。Thermocellum在之前的Discovey赠款中开始。将通过RTqPCR方法以及不同能量分子的相互作用来确定与“葡萄糖”糖酵解相关的关键基因产物的生长条件特异性表达差异。这些数据将导致纤维素发酵的稳健代谢模型。类似的方法将用于C。stercorarium和C. straminivolvens以确认相似性并突出差异。表达模式的变化时，这些生物体相互作用的固体纤维素基质上的混合生物膜将使用蛋白质组学方法进行研究。将使用荧光原位杂交方法观察它们的空间分布。一种假设是C.由于其表面纤维素酶的结构不同，粪石藻可能是浮游性的，而C. thermocellum和C.可以连接两种都携带纤维素体的straminsolvens。这些实验也将使用热自养甲烷热杆菌作为与纤维素降解环境相关的末端氢受体，以及也与C.环境中的热纤维素。通过这项工作产生的代谢理解将导致开发生物体和发酵策略，预计将提高绿色经济的纤维素生物质乙醇生产效率。*

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.*****