Engineering Clostridium saccharoperbutylacetonicum for High Level Isopropanol-Butanol-Ethanol (IBE) Production from Acetic Acid Pretreated Switchgrass Using the CRISPR-Cas9 System

We previously established a biomass pretreatment approach using acetic acid (which can be used as a carbon source for acetone-butanol-ethanol (ABE) fermentation) as the chemical catalyst, leading to comprehensive biomass utilization and enhanced solvent production (especially acetone production due to reassimilation of elevated fatty acids) using Clostridium saccharoperbutylacetonicum. However, acetone is corrosive to engine parts and cannot be used as a fuel. Thus, acetone produced during ABE fermentation is considered as an undesirable byproduct. In this study, we metabolically engineer C. saccharoperbutylacetonicum for isopropanol-butanol-ethanol (IBE) production by introducing secondary alcohol dehydrogenase gene to convert acetone into isopropanol. With either plasmid-based or CRISPR-Cas9-mediated chromosomal-integration-based overexpression, efficient IBE production was achieved. To further enhance solvent production, we additionally overexpressed sol operon (ald-ctfA-ctfB-adc), expression cassette EC (thl-hbd-crt-bcd) or sol in combination with EC. All resultant mutants generated elevated solvents, with one mutant produced 34.2 g/L IBE with a yield of 0.48 g/g. Finally, simultaneous saccharification and fermentation was carried out with the mutant using acetic-acid-pretreated switchgrass, and 16.2 g/L IBE was produced. Our engineered strain produced the highest IBE that has ever been reported in a batch fermentation. Our results indicated that acetic-acid-pretreated biomass can be efficiently converted into biofuel using metabolically engineered Clostridium.