ABSTRACTClostridium cellulovoransDSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, itsn-butanol production from lignocellulosic biomass has yet to be demonstrated. This study demonstrates the construction of a coenzyme A (CoA)-dependent acetone-butanol-ethanol (ABE) pathway inC. cellulovoransby introducingadhE1andctfA-ctfB-adcgenes fromClostridium acetobutylicumATCC 824, which enabled it to producen-butanol using the abundant and low-cost agricultural waste of alkali-extracted, deshelled corn cobs (AECC) as the sole carbon source. Then, a novel adaptive laboratory evolution (ALE) approach was adapted to strengthen then-butanol tolerance ofC. cellulovoransto fully utilize itsn-butanol output potential. To further improven-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as a host for metabolic engineering. Then-butanol production from AECC of the engineeredC. cellulovoranswas increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter. This method represents a milestone towardn-butanol production by CBP, using a single recombinant clostridium strain. The engineered strain offers a promising CBP-enabling microbial chassis forn-butanol fermentation from lignocellulose.IMPORTANCEDue to a lack of genetic tools,Clostridium cellulovoransDSM 743B has not been comprehensively explored as a putative strain platform forn-butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering ofC. cellulovoransfor the development of a CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve then-butanol production ofC. cellulovoransfrom the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). Then-butanol production from AECC was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter, which represents the highest titer ofn-butanol produced using a single recombinant clostridium strain by CBP reported to date. This engineered strain serves as a promising chassis forn-butanol production from lignocellulose by CBP.
Metabolic process engineering ofClostridium tyrobutyricumΔack-adhE2for enhanced n-butanol production from glucose: Effects of methyl viologen on NADH availability, flux distribution, and fermentation kinetics
