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

将发酵途径与木质纤维素梭状芽孢杆菌和相关生物体的能量保存联系起来

• 批准号: RGPIN-2019-05878
• 负责人: Sparling, Richard
• 金额: $ 3.64万
• 依托单位: University of Manitoba
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2021
• 资助国家: 加拿大
• 起止时间: 2021-01-01 至 2022-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=738561
• 关键词: Relating; fermentation; pathways; energy; conservation

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.

基于石油的液体运输燃料的替代品是植物性乙醇。乙醇生产的可发酵糖的丰富，虽然是顽固的，是源自植物生物量（稻草，木材等）的木质纤维素。当前的方法涉及复杂的预处理以生产酵母发酵糖。替代的合并生物处理方法可以使用纤维素发酵梭状芽胞杆菌直接从木质纤维素中产生乙醇。代谢建模一直是一个问题，因为它的“非典型”糖酵解途径，该糖酵解途径与ATP一起使用GTP和Pyrophophate，并且缺乏丙酮酸激酶。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Xylan发酵的C. stercorarium和耐气的纤维素降解C. stramisolvens合作，这些问题可能会解决。然而，从生化上讲，这些后一种生物的特征很差。该计划的目标是了解这三种相关生物中的节能途径。在实现其目标时，该计划将培训2博士学位，2 M.Sc.和5个本科HQP。目标是：a）定义中央代谢途径酶的作用，尤其是在所使用的能量辅助因子（GTP，ATP，焦磷酸）方面，以便更好地了解其独特的代谢作用。 b）了解这些生物的相互作用在木质纤维素作为碳源的共培养中生长时。我们建议完成在先前的盘状授予中，始于热蛋白梭状芽孢杆菌中糖酵解相关的糖酵解酶的表征。与“异型”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由RTQPCR方法以及不同能量分子的相互作用确定。这样的数据将导致纤维素发酵的强大代谢模型。类似的方法将用于史加氏梭菌和杂霉菌，以确认相似性并突出差异。当这些生物作为固体纤维素底物上的混合生物膜相互作用时，表达模式会发生变化。将使用蛋白质组学方法研究。将使用荧光内置杂交方法观察它们的空间分布。一个假设是，胸梭状芽孢杆菌可能是由于其表面纤维素酶的不同结构而具有浮力的，而c. thermocellum和C. stramisolvens则可以携带纤维素。这些实验还将使用甲烷摩托杆菌的热养酸作为与纤维素降解环境以及depilis caldibacillus debilis相关的末端氢受体，这是一种与环境中的辅助毒杆菌相关。通过这项工作产生的代谢理解将导致发展的组织和发酵策略，这些策略有望提高纤维素生物量从绿色经济中提高乙醇生产的效率。