(191bh) Metabolic Engineering of Clostridium cellulovoran for n-Butanol Production from Cellulose

n-Butanol can be used as a significant industrial chemical and potential superior biofuel. Especially, compared with ethanol, n-butanol can be used as an ideal substitute of gasoline because of its high energy density, low water solubility, and low vapor pressure. Unfortunately, bio-butanol production using the conventional acetone-butanol-ethanol (ABE) fermentation process is not economically feasible because of the co-production of other metabolites, including CO₂, acetone, acetate, butyrate, ethanol, and lactate resulting in a low butanol yield. In addition, starch and sugar as subtract are not suitable for the production of bulk biochemicals or biofuels, due to the high feedstock cost and impact on food supply. Lignocellulosic biomass is an abundant, cheap, and renewable source of carbon source, and thus is a desired feedstock for biofuels production. Conventional biorefinery of lignocellulosic biomass requires different operations, including feedstock pretreatment, cellulase/hemicellulase production, biomass enzymatic hydrolysis, sugar fermentation, and product recovery, which is not cost-effective due to the complex process and subsequently high equipment capital requirement. Consolidated bioprocessing (CBP) combines enzyme production, biomass hydrolysis, and sugar fermentation into one step, which will greatly simplify the process and dramatically reduce the equipment investment. In our previous research, Clostridium cellulovorans, a natural cellulose/hemicelluloses utilizing, non-natural butanol producing bacterium, was metabolically engineered for n-butanol production from cellulose by overexpressing adhE2 gene from Clostridium acetobutylicum. However, this mutant only produced 1.42 g/L n-butanol and 1.60 g/L ethanol from microcrystalline cellulose in 10 days. Moreover, due to the plasmid DNAs were always digested by C. cellulovorans cell extract, current transformation efficiency is low, making metabolic engineering difficult. So, efficient transformation for developing better mutants are urgently required. In order to solve this problem, its restriction modification (RM) systems were analyzed and a new clostridium shuttle plasmid p83151d2 without restriction sites was constructed for its successful transformation by improved electroporation in this study. Ethanol and n-butanol production from cellulose were investigated by introduced adhE1, bdhB, aor, thl, fnr, and fdh with adhE2 into C. cellulovorans, respectively. Moreover, adding artificial electron carrier such as methyl viologen (MV) in engineered C. cellulovorans redistributed the metabolic flux from acid production to alcohol production, leading to a high n-butanol titer of 5.2 g/L from cellulose. These results showed that C. cellulovorans is a promising CBP platform host for biofuel production from cellulosic biomass.