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

以植物为基础的乙醇是石油为基础的液体运输燃料的替代品。从植物生物质（秸秆、木材等）中提取的木质纤维素是乙醇生产中大量的可发酵糖的来源。目前的方法包括复杂的预处理，以生产酵母发酵糖。另一种固结生物工艺可以利用纤维素发酵的热胞梭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和耐空气的纤维素降解菌合作，这些问题可能会得到解决。然而，从生物化学的角度来看，后一种生物的特征很差。***该计划的目标是了解这3种相关生物的能量保存途径。为实现其目标，该项目将培养2名博士，2名硕士和5名本科HQP。***目的是：***A)定义中枢代谢途径中酶的作用，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生长时的相互作用。***我们建议完成C. thermocellum中糖酵解的能量守恒相关酶的表征，这是之前的discovery基金开始的。与“非典型”糖酵解相关的关键基因产物的生长条件特异性表达差异将通过RTqPCR方法确定，以及不同能量分子的相互作用。这些数据将导致纤维素发酵的强大代谢模型。将采用类似的方法来确定粪孢弧菌和菌株弧菌的相似性并突出差异。当这些生物作为混合生物膜在固体纤维素基质上相互作用时，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。有一种假说认为，由于C. stercorarium表面纤维素酶的结构不同，它可能是远洋的，而C. thermocellum和C. straminisolvens都携带纤维素，可能会附着在上面。这些实验还将使用热自养甲烷杆菌作为与纤维素降解环境相关的终端氢受体，以及与环境中的热细胞胞杆菌相关的兼性需氧细菌Caldibacillus deweis。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，有望提高绿色经济中纤维素生物质乙醇生产的效率。*****

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.*****