Engineering Clostridium cellulovorans for highly selective n-butanol production from cellulose in consolidated bioprocessing.

Cellulosic n-butanol from renewable lignocellulosic biomass has gained increased interest. Previously, we have engineered Clostridium cellulovorans, a cellulolytic acidogen, to overexpress the bifunctional butyraldehyde/butanol dehydrogenase gene adhE2 from C. acetobutylicum for n-butanol production from crystalline cellulose. However, butanol production by this engineered strain had a relatively low yield of approximately 0.22 g/g cellulose due to the coproduction of ethanol and acids. We hypothesized that strengthening the carbon flux through the central butyryl-CoA biosynthesis pathway and increasing intracellular NADH availability in C. cellulovorans adhE2 would enhance n-butanol production. In this study, thiolase (thlA ) from C. acetobutylicum and 3-hydroxybutyryl-CoA dehydrogenase (hbd ) from C. tyrobutyricum 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. Metabolic flux analyses showed that mutants overexpressing these genes had a significantly increased carbon flux toward butyryl-CoA, which resulted in increased production of butyrate and butanol. The addition of methyl viologen as an electron carrier in batch fermentation further directed more carbon flux towards n-butanol biosynthesis due to increased reducing equivalent or NADH. The engineered strain C. cellulovorans adhE2-fnr -thlA -hbd produced n-butanol from cellulose at a 50% higher yield (0.34 g/g), the highest ever obtained in batch fermentation by any known bacterial strain. The engineered C. cellulovorans is thus a promising host for n-butanol production from cellulosic biomass in consolidated bioprocessing.