(228ad) Rationally Metabolic Engineering of Clostridium Cellulovorans for Butanol Production

Clostridium cellulovorans can produce butanol from biomass through consolidated bioprocessing. In this study, we rationally metabolically engineered the C. cellulovorans to produce lignocellulosic butanol. Our proteomics analysis showed that the high expression of thiolase (thl), which catalyzes the reaction from acetyl-CoA to acetoacetyl-CoA, had strong correlation with butanol production, so the heterologous thl gene from C. tyrobutyricum was overexpressed in C. cellulovorans. To selectively produce butanol, the aldehyde dehydrogenase (ald) and butanol dehydrogenase (bdh) were synthesized to covert butyryldehyde to butanol. The butanol production is conventionally limited by the byproduct formation during the fermentation, such as acetate. ClosTron-mediated engineering and CRISPR interference technology were applied to decrease the acetate kinase and phosphotransacetylase activities, which are directly related to acetate formation. Finally, the constructed engineered mutants of C. cellulovorans were evaluated in consolidated bioprocessing. The acetate was decrease by > 50% and the selectivity of butanol was improved to >0.5 g/g. The lignocellulosic fermentation produced butanol with titer of >2 g/L with the yield at 0.2g/g. The changes in carbon, energy and redox balance in response to metabolic engineering were also identified to assist further engineering of butanol production process.