Improved n-butanol production from Clostridium cellulovorans by integrated metabolic and evolutionary engineering

Clostridium cellulovorans DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its n-butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a CoA-dependent acetone-butanol-ethanol (ABE) pathway in C. cellulovorans by introducing genes of adhE1 and ctfA-ctfB-adc from C. acetobutylicum ATCC 824, which enabled it to produce n-butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen the n-butanol tolerance of C. cellulovorans , to fully utilize its n-butanol output potential. To further improve n-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as host for metabolic engineering. The n-butanol production from AECC of the engineered C. cellulovorans enhanced 138-fold from less than 0.025 g/L to 3.47 g/L. This method represents a milestone toward n-butanol production by CBP, using single recombinant clostridia. The engineered strain offers a promising CBP-enabling microbial chassis for n-butanol fermentation from lignocellulose. Importance Due to a lack of genetic tools, Clostridium cellulovorans DSM 743B has not been comprehensively explored as a putative strain platform for n-butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering of C. cellulovorans for the development of CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve the n-butanol production of C. cellulovorans from the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). The n-butanol production from AECC enhanced by 138-fold from less than 0.025 g/L to 3.47 g/L, which represents the highest titer of n-butanol produced using single recombinant clostridia by CBP reported to date. This engineered strain serves as a promising n-butanol production chassis from lignocellulose by CBP.