(176ad) Improving Butanol Tolerance of C. Tyrobutyricum By Introducing Heat Shock Proteins from an Extremophilic Bacterium

Butanol, a four-carbon aliphatic saturated alcohol, is a promising drop-in biofuel with excellent fuel properties and similar energy density to gasoline. Commercial production of biobutanol in Acetone-Butanol-Ethanol (ABE) fermentation with solventogenic clostridia such as Clostridium acetobutylicum and Clostridium beijerinckii, which is difficult to control or manipulate because of their complicated metabolic pathways and life cycle. Recently, Clostridium tyrobutyricum, a Gram-positive, spore-forming, obligate anaerobic hyper-butyrate producer, was engineered to produce butanol from lignocellulosic biomass hydrolysate sugars. However, the metabolic products including butanol and organic acids, and the lignocellulosic hydrolysate-derived inhibitors like furfural and phenolics strongly inhibited cell growth and butanol production, leading to low butanol titer, production rate and yield. Therefore, improving tolerance of C. tyrobutyricum to butanol and hydrolysate inhibitors is necessary for economical production of biobutanol. Heat shock proteins (HSPs), which could prevent protein aggregation and assist in protein folding and refolding, play important roles in bacterial stress response. They can be induced by various environmental stresses including temperature, acid, solvent, osmotic pressure and drying. In this study, heat shock proteins such as GroESL and DnaK known to increase resistance to lignocellulosic inhibitory compounds were overexpressed in C. tyrobutyricum. Specifically, we introduced the HSPs GroESL and DnaK from an extremely radioresistant bacterium Deinococcus wulumuqiensis R12 into C. tyrobutyricum ATCC 25755 to improve its robustness and tolerance to butanol, resulting in mutant strains that produced butanol at >20 g/L from corn stover hydrolysate, an over 50% improvement compared to the control strains, in batch fermentation.