(188ao) Improved n-Butanol Production of Clostridium Cellulovorans by Integrated Metabolic, Evolution and Process Engineering

Clostridium cellulovorans DSM 743B has potential as chassis strain for biomass refinery by consolidated bioprocessing (CBP). However, its comprehensive strain engineering has yet to be demonstrated. In the study, we constructed a CoA-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing genes of adhE1 and ctfA-ctfB-adc from Clostridium acetobutylicum ATCC 824, which enabled it to produce n-butanol directly with alkali-extracted, deshelled corn cobs (AECC), an abundant and low-cost agricultural waste, as sole carbon source. Then a laboratory adaptive evolution was implemented to strengthen C. cellulovorans’ n-butanol tolerance, and ultimately enhanced n-butanol output. Finally, a two-stage pH control strategy was adapted to improve n-butanol production further. The engineered C. cellulovorans’ n-butanol production from AECC enhanced by 138-fold from less than 0.025 g/L to 3.47 g/L. Our exploration represented a milestone towards n-butanol production by CBP using single microorganism. The developed C. cellulovorans serves as a promising CBP-enabling microbial chassis for n-butanol fermentation from lignocellulose.