Molecular inactivation of exopolysaccharide biosynthesis inPaenibacillus polymyxaDSM 365 for enhanced 2,3-butanediol production

Formation of Exopolysaccharides (EPS) during 2,3-butanediol (2,3-BD) fermentation byPaenibacillus polymyxadecreases 2,3-BD yield, increases medium viscosity and impacts 2,3-BD downstream processing. Therefore, additional purification steps are required to rid the fermentation broth of EPS prior to 2,3-BD purification, which adds to the production cost. To eliminate EPS production during 2,3-BD fermentation, we explored a metabolic engineering strategy to disable the EPS production pathway ofP. polymyxa, thereby increasing 2,3-BD yield and productivity. The levansucrase gene which encodes levansucrase, the enzyme responsible for EPS biosynthesis inP. polymyxa, was successfully disrupted. The resultingP. polymyxalevansucrase null mutant showed 34% and 54% increases in growth with 6.4- and 2.4-folds decrease in EPS formation in sucrose and glucose cultures, respectively. The observed decrease in EPS formation by the levansucrase null mutant may account for the 27% and 4% increase in 2,3-BD yield, and 4% and 128% increases in 2,3-BD productivity when grown on sucrose and glucose media, respectively. Genetic stability of the levansucrase null mutant was further evaluated. Interestingly, the levansucrase null mutant remained genetically stable over fifty generations with no observable decrease in growth and 2,3- BD formation with or without antibiotic supplementations. Collectively, our results show thatP. polymyxalevansucrase null mutant has potential for improving 2,3-BD yield, and ultimately, the economics of large-scale microbial 2,3-BD production.