Improved <i>n</i> -Butanol Production from Clostridium cellulovorans by Integrated Metabolic and Evolutionary Engineering

Abstract

<i>Clostridium cellulovorans</i> DSM 743B offers potential as a chassis strain for biomass refining by consolidated bioprocessing (CBP). However, its <i>n</i>-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 in <i>C. cellulovorans</i> by introducing <i>adhE1</i> and <i>ctfA-ctfB-adc</i> genes from <i>Clostridium acetobutylicum</i> ATCC 824, which enabled it to produce <i>n</i>-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 the <i>n</i>-butanol tolerance of <i>C. cellulovorans</i> to fully utilize its <i>n</i>-butanol output potential. To further improve <i>n</i>-butanol production, both metabolic engineering and evolutionary engineering were combined, using the evolved strain as a host for metabolic engineering. The <i>n</i>-butanol production from AECC of the engineered <i>C. cellulovorans</i> was increased 138-fold, from less than 0.025 g/liter to 3.47 g/liter. This method represents a milestone toward <i>n</i>-butanol production by CBP, using a single recombinant clostridium strain. The engineered strain offers a promising CBP-enabling microbial chassis for <i>n</i>-butanol fermentation from lignocellulose.<b>IMPORTANCE</b> Due to a lack of genetic tools, <i>Clostridium cellulovorans</i> DSM 743B has not been comprehensively explored as a putative strain platform for <i>n</i>-butanol production by consolidated bioprocessing (CBP). Based on the previous study of genetic tools, strain engineering of <i>C. cellulovorans</i> for the development of a CBP-enabling microbial chassis was demonstrated in this study. Metabolic engineering and evolutionary engineering were integrated to improve the <i>n</i>-butanol production of <i>C. cellulovorans</i> from the low-cost renewable agricultural waste of alkali-extracted, deshelled corn cobs (AECC). The <i>n</i>-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 of <i>n</i>-butanol produced using a single recombinant clostridium strain by CBP reported to date. This engineered strain serves as a promising chassis for <i>n</i>-butanol production from lignocellulose by CBP.