Enhancing butanol tolerance in Solventogenic Clostridia Species - Structural and Functional Investigation

环境友好的生物质燃料是利用化石燃料等不可再生能源生产能源的一种有吸引力的替代方案。生物丁醇是一种很有前途的新一代生物燃料和/或生物燃料添加剂，因为它具有高能量含量和燃烧效率，与汽油具有良好的调和性能，不需要发动机和炼油厂改装，运输安全。产溶剂型梭状芽孢杆菌是一种厌氧微生物，它利用将碳水化合物转化为丙酮、丁醇和乙醇的代谢途径。这些溶剂发酵作为石油化工生产丙酮和丁醇的一种可行和经济的竞争对手，引起了人们的极大兴趣。然而，尽管对控制这一代谢途径的单个基因进行了鉴定和表征，但细胞丁醇感知机制以及促进生存和生长的潜在适应机制仍然知之甚少。溶剂型梭状芽孢杆菌对高浓度丁醇的低耐受性仍然是工业生产丁醇的主要障碍之一。最近，一项比较转录分析显示，一个保守的基因簇在丁醇胁迫下显著上调。已识别的丁醇耐受相关基因转录一个三磷酸腺苷(ATP)结合盒输出器，旁边是一个调节的双组分系统(TCS)。在丁醇胁迫条件下，这些基因簇转录的蛋白质的过表达导致生长速度和溶剂产量的增加。因此，对该基因模块进行深入的生化和结构特征分析，是构建能耐受工业规模发酵过程中不断增加的丁醇胁迫的溶剂型梭状芽孢杆菌改良菌株的有效途径。本研究旨在利用先进的生化和结构生物学方法，包括低温电子显微镜和X射线结晶学，对新型丁醇耐受相关基因簇产物的结构-功能关系进行表征。对这一模块的详细理解将在原子水平上阐明在丁醇应激下促进生存和生长的潜在适应机制。这一研究方案的结果将作为合理设计高效工业发酵的强化菌株的基石。反过来，这些改良菌株将对利用可再生资源、质量优于丁醇化学生产的生物丁醇行业产生深远影响。我们的发现也将促进对调控ATP结合盒出口商和调控TCS对的分子机制的了解。最后，使用多种尖端科学技术将使我的受训人员掌握与学术和工业环境相关的宝贵技能，为加拿大的知识经济做出贡献。

Environmentally friendly biomass-produced fuels are an attractive alternative for energy production from unrenewable sources, such as fossil fuels. Biobutanol is a promising next-generation biofuel and/or biofuel additive due to its high energy content and burning efficiency, excellent blending properties with gasoline, does not require engine and refinery modifications and is safe to transport. Solventogenic clostridia species are anaerobic microorganisms that employ a metabolic pathway converting carbohydrates into acetone, butanol, and ethanol. These solvents fermentation attract great interest as a viable and economic competitor to the production of acetone and butanol by the petrochemical industries. However, despite the identification and characterization of the individual genes governing this metabolic pathway, the cellular butanol sensing mechanisms, along with the underlying adaptive mechanisms, promoting survival and growth, remain poorly understood. The low tolerance of solventogenic clostridia species to high butanol concentrations remains one of the major hurdles for butanol production at the industrial level. Recently, a comparative transcriptomic analysis revealed a significant upregulation of a conserved genes cluster in response to butanol stress. The identified butanol tolerance-related genes transcribe an adenosine-triphosphate (ATP) binding cassette exporter alongside a regulatory two-component system (TCS). Overexpression of the proteins transcribed by these genes cluster resulted in increased growth rate and solvent production under butanol stress conditions. Therefore, an in-depth biochemical and structural characterization of this gene module is a promising avenue for engineering improved solventogenic clostridia strains that can endure the increasing butanol stress during industrial-scale fermentation. This research proposal aims to characterize the structure-function relationship of the novel butanol tolerance-related gene cluster products by advanced biochemical and structural biology methods, including cryo-electron microscopy and X-ray crystallography. A detailed understanding of this module will shed light on the underlying adaptive mechanisms promoting survival and growth under butanol stress at the atomic level. Results obtained from this research proposal will serve as the cornerstone for the rational design of enhanced strains for efficient industrial fermentation. In turn, these improved strains will have a profound impact on the biobutanol industry that utilizes renewable resources and is superior in quality to the chemical production of butanol. Our findings will also advance the knowledge of the molecular mechanisms governing ATP binding cassette exporters and regulatory TCS pairs. Finally, the use of multiple cutting-edge scientific techniques will equip my trainees with valuable skills relevant for both academic and industrial settings, contributing to the knowledge-based economy of Canada.