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

我一直在工作（加拿大基因组项目，结束日期9月。2014），其中厌氧细菌能够将林业和农业植物废弃物生物质转化为生物燃料，例如乙醇和氢气，主要是热硫化氢嗜热厌氧杆菌（Thermoanaerostriumthermohydrosulfuricus）和热纤梭菌（Clostridiumthermocellum）。它们将纤维素和/或半纤维素降解为乙醇的能力使得这些“感兴趣的生物体”用于从这些类型的废弃生物质生产生物燃料。然而，流向乙醇的碳和电子与产生不太理想的最终产品的发酵分支竞争。这些生物的基因组序列是已知的。然而，一些关键基因的产物已经被生物化学表征，并且主要基于序列同源性提出功能分配。此外，一些预期的反应似乎缺乏或非常规。我的小组对这些生物体进行的蛋白质组学和转录组学（'omic）分析表明，在测试的生长条件下，几种基因产物要么过度表达，要么表达不足。 这导致了关于参与该生物质组分发酵的关键基因产物和辅因子的实际功能的问题。这些知识对于基因工程的准确建模和合理目标选择至关重要，以提高纤维素的乙醇产量。基于组学分析，NSERC Discovery研究计划提出研究这些生物体中发酵途径和节能的整合。利用表达载体在E.在大肠杆菌中，将纯化对应于推定的ATP和焦磷酸盐依赖性分解代谢基因的蛋白质，并表征以确认酶功能和辅因子使用。与我的学生一起，我还将研究特定基因敲除对碳和能量流动的影响，以及对特定发酵分支中涉及的蛋白质表达的调节。为此，T。将使用热硫化氢，因为它适合于基因插入和敲除技术的开发。该生物体的基因组含有与不同类型的氢化酶、膜-和可溶性PPi酶、ATP和PPi依赖性丙酮酸二激酶和磷酸果糖激酶以及可能参与生物质转化为乙醇和氢的多种醇脱氢酶一致的序列。这种明显的功能冗余使其成为一种有用的模式生物，在其中使用敲除突变体来研究这些酶的相对重要性。然而，进行此类实验的第一步是根据为其他热厌氧杆菌开发的程序开发一个遗传系统。我们还没有能够观察到在这些生物体中完全发酵为氢气+CO2+乙酸盐，最有可能是因为培养基中的氢气过饱和。因此，我们没有观察到预期与这些生物体中的任一种的氢产生相关的关键酶的显著表达，例如质子易位（Ech）氢化酶或钠易位RNF-like铁氧还蛋白-NADH氧化还原酶。在成功地分析了来自共培养物的混合转录组后，我们建议在除氢的Methanothermobacter thermoautotrophicus存在下的纤维二糖发酵过程中进行蛋白质组学分析。预计这将推动我们感兴趣的生物体的氢气生产，并提供对相对于乙醇和其他竞争性发酵产物产生氢气的发酵分支的调节的见解。在拟议的研究计划结束时，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，这些都是掌握进一步就业的重要工具。

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.