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

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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.