Metabolic Engineering of <i>Clostridium cellulovorans</i> to Improve Butanol Production by Consolidated Bioprocessing

Abstract

<i>Clostridium cellulovorans</i> DSM 743B can produce butyrate when grown on lignocellulose, but it can hardly synthesize butanol. In a previous study, <i>C. cellulovorans</i> was successfully engineered to switch the metabolism from butyryl-CoA to butanol by overexpressing an alcohol aldehyde dehydrogenase gene <i>adhE1</i> from <i>Clostridium acetobutylicum</i> ATCC 824; however, its full potential in butanol production is still unexplored. In the study, a metabolic engineering approach based on a push-pull strategy was developed to further enhance cellulosic butanol production. In order to accomplish this, the carbon flux from acetyl-CoA to butyryl-CoA was pulled by overexpressing a trans-enoyl-coenzyme A reductase gene (<i>ter</i>), which can irreversibly catalyze crotonyl-CoA to butyryl-CoA. Then an acid reassimilation pathway uncoupled with acetone production was introduced to redirect the carbon flow from butyrate and acetate toward butyryl-CoA. Finally, xylose metabolism engineering was implemented by inactivating <i>xylR</i> (<i>Clocel_0594</i>) and <i>araR</i> (<i>Clocel_1253</i>), as well as overexpressing <i>xylT</i> (<i>CA_C1345</i>), which is expected to supply additional carbon and reducing power for CoA and butanol synthesis pathways. The final engineered strain produced 4.96 g/L of <i>n</i>-butanol from alkali extracted corn cobs (AECC), increasing by 235-fold compared to that of the wild type. It serves as a promising butanol producer by consolidated bioprocessing.