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

我一直在研究（Genome Canada project，截止日期2014年9月）能够将林业和农业植物废弃物转化为乙醇和氢气等生物燃料的厌氧细菌，主要是热厌氧菌thermohydrosulphur和Clostridium thermocellum。它们将纤维素和/或半纤维素降解为乙醇的能力使这些“感兴趣的生物”从这些类型的废弃生物质中生产生物燃料。然而，流向乙醇的碳和电子与产生不太理想的最终产物的发酵分支竞争。这些生物体的基因组序列是已知的。然而，很少有关键基因的产物得到了生物化学表征，功能定位主要是基于序列同源性。此外，一些预期的反应似乎缺乏或非传统。我的团队对这些生物体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，一些基因产物要么过度表达，要么过低表达。这就引出了关于关键基因产物和辅助因子的实际功能的问题，这些基因产物和辅助因子参与了这种生物质成分的发酵。这些知识对于精确建模和合理选择基因工程目标以提高纤维素乙醇产量至关重要。**基于组学分析，这个NSERC发现研究计划提出在这些生物中研究发酵途径和能量节约的整合。利用大肠杆菌中的表达载体，将纯化和表征与假定的ATP和焦磷酸依赖分解代谢基因相对应的蛋白质，以确定酶的功能和辅助因子的使用。与我的学生一起，我还将研究特定基因敲除对碳和能量流动的影响，以及对特定发酵分支中蛋白质表达的调节。为此，热氢硫霉将被使用，因为它适合于基因插入和敲除技术的发展。该生物的基因组包含与不同类型的氢化酶、膜溶性和可溶性PPiase、ATP和PPi依赖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的多种醇脱氢酶相一致的序列。这种明显的功能冗余使其成为一种有用的模式生物，在这种模式生物中，使用敲除突变体研究这些酶的相对重要性。然而，实现这类实验的第一步是在为其他热厌氧菌开发的程序的基础上开发一个遗传系统。**我们未能观察到在这些生物中只发酵成氢+ CO2 +醋酸盐，很可能是因为培养基中的氢过饱和。因此，我们在这两种生物中都没有观察到与产氢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样铁氧化还蛋白- nadh氧化还原酶。在成功地分析了共同培养的混合转录组后，我们建议在脱氢热自养甲烷菌存在的情况下，在纤维素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，并提供对导致氢气相对于乙醇和其他竞争发酵产物的发酵分支的调控的见解。在计划的研究项目结束时，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.