(712e) Comparative Proteomics Analysis of High Butyrate and Butanol Producing Clostridium Tyrobutyricum Mutants

As a sustainable and environmentally friendly biofuel, biobutanol is an excellent potentially substitute for gasoline without any engine modification. Metabolic engineering has been applied in Clostridium tyrobutyricum and shifted carbon from butyrate in ACKKO strain to butanol in ACKKO-adhE2 strain. To further increase the production of butanol through further metabolic engineering, we applied comparative proteomics to evaluate the host cell protein expression in response to butyrate and butanol rebalance. First, the 2 L free-cell fermentations were performed at pH 6.0 and 37 ^oC to compare the butanol and butyrate productions among wild type, ACKKO mutant, and ACKKO-adhE2 mutant. A high butanol titer of 16 g/L was obtained by ACKKO-adhE2, and high butyric acid titer of 37 g/L was achieved by ACKKO. Second, the global proteomics map of more than 3000 proteins in these three strains collected from bioreactor were analyzed and compared. The proteomics data revealed significant change in the key enzymes related to acid and solvent end products, central pathway, redox balance, energy and cell growth. It is found that both carbon balance and redox balance were critical to regulate butanol production. Third, the preliminary results of intracellular and extracellular metabolite analysis also identified the butanol formation related amino acids and fatty acids. Overall, the integration of comparative proteomics study assisted by metabolite analysis with fermentation with accurate process control provided us an in-depth fundamental understanding of C4 (i.e. butyrate and butanol) production regulation. These findings are being used to further increase butanol production via metabolic cell-process engineering.