Integration of fermentation and energy conservation pathways in thermophilic, lignocellulolytic clostridia and related anaerobes.

嗜热、木质纤维素梭菌和相关厌氧菌中发酵和节能途径的整合。

• 批准号: RGPIN-2014-06173
• 负责人: Sparling, Richard
• 金额: $ 2.55万
• 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: 2014
• 资助国家: 加拿大
• 起止时间: 2014-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=567638
• 关键词: Integration; fermentation; energy; conservation; pathways

I have been working (Genome Canada project, end date Sept. 2014) with anaerobic bacteria capable of converting forestry and agricultural plant-waste biomass into biofuels such as ethanol and hydrogen, mainly Thermoanaerobacter thermohydrosulfuricus and Clostridium thermocellum. Their ability to degrade cellulose and/or hemicellulose to ethanol has made these “organisms-of-interest” for biofuels production from these types of waste biomass. However, the flow carbon and electrons to ethanol competes with fermentation branches that produce less desirable end-products. The genome sequence of these organisms is known. However, the products of few key genes have been characterized biochemically, and the assignment of function mainly being proposed on the basis of sequence homology. Also, some expected reactions appear to be lacking or non-conventional. Proteomic and transcriptomic ('omic) analyses performed by my group for these organisms have shown that several gene products are either over- or under-expressed under the growth conditions tested. This leads to questions as to the actual function of key gene products and co-factors involved in the fermentation of components of this biomass. Such knowledge is crucial for accurate modelling and rational target selection for genetic engineering to improved ethanol yields from cellulose. Based on 'omics analyses, this NSERC Discovery research program proposes to study the integration of fermentation pathways and energy conservation in these organisms. Using expression vectors in E. coli, proteins corresponding to putative ATP and pyrophosphate dependent catabolic genes will be purified and characterized to confirm enzymatic function and co-factor use. Together with my students, I will also study the effect of specific gene knockouts on the flow of carbon and energy, as well as on the regulation of protein expression involved in specific fermentative branches. For this, T. thermohydrosulfuricus will be used since it is amenable for the development of techniques for gene insertion and knockout. This organism’s genome contains sequences consistent with different types of hydrogenases, membrane- and soluble PPiase, ATP and PPi dependent pyruvate dikinase and –phosphofructokinase, as well as multiple alcohol dehydrogenases that may be involved in the conversion of the biomass to ethanol and hydrogen. This apparent functional redundancy makes this a useful model organism in which to study the relative importance of these enzymes using knock-out mutants. Nevertheless, the first step towards such experiments is to develop a genetic system based on procedures developed for other Thermoanaerobacters. We have not been able to observe fermentation to exclusively hydrogen plus CO2 plus acetate in these organisms, most likely because of hydrogen supersaturation of the medium. As a consequence, we have not observed significant expression of key enzymes expected to be aaociated with hydrogen production in either of these organisms, for example the proton-translocating (Ech) hydrogenase or sodium translocating RNF-like ferredoxin-NADH oxidoreductase. Having had success with the analysis of mixed transcriptomes from co-cultures, we propose to perform proteomic analyses during cellobiose fermentation in the presence of hydrogen removing Methanothermobacter thermoautotrophicus. This is expected to drive hydrogen production from our organisms of interest, and provide insights into the regulation of the fermentation branches leading to hydrogen relative to ethanol and other competing fermentation products. At the end of the proposed research program, my students will have gained expertise in biochemisty, molecular biology and bioinformatics, which are all important tools to master for further employment.

我一直在工作（加拿大基因组项目，2014年9月结束），厌氧菌细菌能够将林业和农业植物废物生物量转化为生物燃料，例如乙醇和氢，主要是热虫杆菌热氢硫硫硫硫酸硫酸属菌和热themocellum。它们将纤维素和/或半纤维素降解为乙醇的能力使这些“生物含量”从这些类型的废物生物量中产生。但是，流向乙醇的流碳和电子与发酵分支竞争，发酵分支产生较少理想的最终产物。这些生物的基因组序列是已知的。但是，很少有关键基因的产物在生化上是表征的，并且主要基于序列同源性提出的功能分配。同样，一些预期的反应似乎缺乏或非规定。我组对这些生物进行的蛋白质组学和转录组（'OMIC）分析表明，在测试的生长条件下，几种基因产物要么过表达或表达不足。这导致了有关关键基因产物的实际功能和参与该生物量成分发酵的共同因素的实际功能。这种知识对于基因工程的准确建模和合理目标选择至关重要，以改善纤维素的乙醇产量。基于“ OMICS分析”，该NSERC发现研究计划的提案建议使用大肠杆菌中的表达向量进行研究，将纯化并表征与假定ATP和焦磷酸分解代谢基因相对应的蛋白质，以确认酶促功能和cofactor的使用。与我的学生一起，我还将研究特定基因敲除对碳和能量流的影响，以及调节特定发酵分支中涉及的蛋白质表达。为此，将使用Themoydrosulfuricus链球菌，因为它适合开发基因插入和敲除技术。该生物体的基因组包含与不同类型的氢化酶，膜和可溶性PPIASE，ATP和PPI依赖性丙酮酸二基酶以及 - 磷酸果糖激酶以及多种酒精脱氢酶，以及可能与生物量转化为乙醇和氢基因的多种酒精脱氢酶。这种明显的功能冗余使这是一种有用的模型生物，在该模型中，使用敲除突变体研究这些酶的相对重要性。然而，迈向这样的实验的第一步是基于针对其他热虫杆菌开发的程序开发遗传系统。我们无法观察到在这些生物体中专门氢加二氧化碳加乙酸二氧化碳加乙酸盐的发酵，这很可能是由于培养基的氢过饱和。因此，我们尚未观察到这些生物中的任何一个预计将与氢生产相关的关键酶的显着表达，例如质子转移（ECH）氢化酶或钠易位RNF样的RNF样氧化还蛋白 - 氧化还蛋白 - 氧化物 - 氧化物 - 氧化还原酶。在分析共培养的混合转录组方面取得了成功，我们建议在氢除去甲基热杆菌热养生的情况下在纤维酶发酵过程中进行蛋白质组学分析。预计这将从我们感兴趣的组织中推动氢生产，并提供有关相对于乙醇和其他竞争发酵产品的发酵分支的调节的见解。在拟议的研究计划的结尾，我的学生将获得生物化学，分子生物学和生物信息学方面的专业知识，这都是掌握进一步就业的重要工具。