(185h) Metabolic engineering of Clostridium cellulovorans for selective n-butanol production from cellulose

To eliminate expensive saccharifying enzyme addition and reduce capital investment, consolidated bioprocessing has been considered as a promising approach for cellulosic butanol production, which combines hydrolytic enzyme production, cellulose/hemicellulose saccharification, and fermentation into one process step using cellulolytic and butanol-producing microorganism or microbial consortia. In our previous research, Clostridium cellulovorans, a natural cellulose/hemicelluloses utilizing, non-natural butanol producing bacterium, was engineered for n-butanol production from cellulose by overexpressing adhE2 gene from Clostridium acetobutylicum. However, this engineered strain only produced 1.42 g/L n-butanol with a relatively low yield of 0.19 g/g cellulose due to the co-production of ethanol and acids. In this study, heterologous thiolase (thlA^CA) and 3-hydroxybutyryl-CoA dehydrogenase (hbd^CT) were overexpressed in C. cellulovorans adhE2 to increase the flux from acetyl-CoA to butyryl-CoA. In addition, ferredoxin-NAD(P)⁺ oxidoreductase (fnr), which can regenerate the intracellular NAD(P)H and thus increase butanol biosynthesis, was also overexpressed. In batch fermentation with methyl viologen as an electron carrier, the engineered strain C. cellulovorans adhE2-fnr^CA-thlA^CA-hbd^CT was able to direct the carbon flux towards n-butanol biosynthesis, leading to a high n-butanol titer of >5 g/L with a high yield of 0.34 g/g cellulose, which were the highest ever obtained for butanol production from cellulose by a bacterial strain. Consolidated bioprocess by engineered C. cellulovorans is thus a promising strategy for bio-butanol production from cellulosic biomass.