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

我一直在工作(Genome Canada项目，结束日期9月有能力将林业和农业植物废弃物生物质转化为乙醇和氢气等生物燃料的厌氧细菌，主要是高温厌氧细菌热氢硫化杆菌和热细胞梭菌。它们将纤维素和/或半纤维素降解成乙醇的能力，使这些生物成为从这些类型的废弃生物质中生产生物燃料的感兴趣的生物。然而，流向乙醇的碳和电子与发酵分支竞争，后者产生的最终产品不那么令人满意。这些生物的基因组序列是已知的。然而，少数关键基因的产物已经被生化鉴定，功能的分配主要是基于序列的同源性。此外，一些意料之中的反应似乎缺乏或非常规。我的团队对这些生物进行的蛋白质组学和转录组学分析表明，在测试的生长条件下，几种基因产物要么过度表达，要么过度表达。这导致了关键基因产物的实际功能和参与这种生物质成分发酵的辅助因子的问题。这些知识对于基因工程提高纤维素乙醇产量的精确建模和合理目标选择至关重要。**基于组学分析，NSERC Discovery研究计划建议研究这些生物中发酵途径和能量守恒的整合。利用在大肠杆菌中的表达载体，与可能依赖于ATP和焦磷酸盐的分解代谢基因相对应的蛋白质将被纯化和鉴定，以确认酶功能和辅因子的使用。我还将与我的学生一起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，将使用热氢硫杆菌，因为它适合于基因插入和敲除技术的发展。这种生物的基因组包含与不同类型氢酶一致的序列，膜和可溶性PPiase，依赖于ATP和PPI的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气的多种酒精脱氢酶。这种明显的功能冗余使其成为利用基因敲除突变体研究这些酶的相对重要性的有用模式生物。然而，这些实验的第一步是在为其他高温厌氧细菌开发的程序的基础上开发一个遗传系统。**我们还没有能够在这些生物中观察到完全由氢+二氧化碳+醋酸盐发酵，很可能是因为介质的氢过饱和。因此，我们没有观察到与产氢有关的关键酶在这两种生物中的显著表达，例如质子转移(Ech)氢酶或钠转移RNF样铁还蛋白-NADH氧化还原酶。在成功分析了混合培养物中的混合转录本后，我们建议在纤维二糖发酵过程中进行蛋白质组学分析，其中存在脱氢甲烷热自养杆菌。这有望推动我们感兴趣的生物体产生氢气，并为导致氢气的发酵分支相对于乙醇和其他竞争发酵产品的调节提供洞察力。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，这些都是为进一步就业而需要掌握的重要工具。

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.