Enhanced solvent production by metabolic engineering of a twin-clostridial consortium

Abstract The efficient fermentative production of solvents (acetone, n -butanol, and ethanol) from a lignocellulosic feedstock using a single process microorganism has yet to be demonstrated. Herein, we developed a consolidated bioprocessing (CBP) based on a twin-clostridial consortium composed of Clostridium cellulovorans and Clostridium beijerinckii capable of producing cellulosic butanol from alkali-extracted, deshelled corn cobs (AECC). To accomplish this a genetic system was developed for C. cellulovorans and used to knock out the genes encoding acetate kinase ( Clocel_1892 ) and lactate dehydrogenase ( Clocel_1533 ), and to overexpress the gene encoding butyrate kinase ( Clocel_3674 ), thereby pulling carbon flux towards butyrate production. In parallel, to enhance ethanol production, the expression of a putative hydrogenase gene ( Clocel_2243 ) was down-regulated using CRISPR interference (CRISPRi). Simultaneously, genes involved in organic acids reassimilation ( ctfAB, cbei_3833/3834 ) and pentose utilization ( xylR, cbei_2385 and xylT, cbei_0109 ) were engineered in C. beijerinckii to enhance solvent production. The engineered twin-clostridia consortium was shown to decompose 83.2 g/L of AECC and produce 22.1 g/L of solvents (4.25 g/L acetone, 11.5 g/L butanol and 6.37 g/L ethanol). This titer of acetone-butanol-ethanol (ABE) approximates to that achieved from a starchy feedstock. The developed twin-clostridial consortium serves as a promising platform for ABE fermentation from lignocellulose by CBP.