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

增强产溶剂梭菌物种的丁醇耐受性 - 结构和功能研究

• 批准号: RGPIN-2022-03031
• 负责人: Zeytuni, Natalie
• 金额: $ 2.7万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2022
• 资助国家: 加拿大
• 起止时间: 2022-01-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=743463
• 关键词: Enhancing; butanol; tolerance; Solventogenic; Clostridia

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.

对环境友好的生物质燃料是一种有吸引力的替代能源生产的不可再生资源，如化石燃料。生物丁醇是一种很有前途的下一代生物燃料和/或生物燃料添加剂，因为它具有高能量含量和燃烧效率，与汽油具有良好的混合性能，不需要对发动机和炼油厂进行改装，并且运输安全。溶剂型梭菌是厌氧微生物，通过代谢途径将碳水化合物转化为丙酮、丁醇和乙醇。这些溶剂发酵作为丙酮和丁醇生产的可行和经济的竞争对手引起了石油化工工业的极大兴趣。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。溶剂型梭菌对高浓度丁醇的低耐受性仍然是工业丁醇生产的主要障碍之一。最近，一项比较转录组学分析揭示了一个保守基因簇在丁醇胁迫下的显著上调。鉴定的丁醇耐受性相关基因转录了腺苷-三磷酸（ATP）结合盒输出器以及调节双组分系统（TCS）。在丁醇胁迫条件下，这些基因簇转录的蛋白过表达导致生长速度和溶剂产量增加。因此，对该基因模块进行深入的生化和结构表征是工程改进溶剂型梭菌菌株的有希望的途径，这些菌株可以在工业规模的发酵过程中承受日益增加的丁醇压力。本研究计划旨在利用先进的生物化学和结构生物学方法，包括冷冻电子显微镜和x射线晶体学，表征新型丁醇耐受性相关基因簇产物的结构-功能关系。对该模块的详细了解将在原子水平上揭示促进丁醇胁迫下生存和生长的潜在适应机制。本研究结果将为合理设计高效工业发酵强化菌株奠定基础。反过来，这些改良菌株将对利用可再生资源、质量优于化学丁醇生产的生物丁醇工业产生深远影响。我们的发现也将促进对ATP结合盒出口和调节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学员具备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。