(697b) Biobutanol Production By C. Tyrobutyricum through Proteomics Guided Carbon and Redox Rebalance

With similar energy intensity, biobutanol is an environmentally friendly substitute for fossil fuel. Clostridium tyrobutyricum is an excellent host cell that can be engineered to produce butanol because of its high butanol tolerance and conversion yield from various rate to butyryl-CoA. Our comparative proteomics study revealed the limiting steps in the central carbohydrate pathway of C. tyrobutyricum. In this study, the rational host cell engineering was performed to redistribute the metabolic flux of carbon and redox to produce butanol. The heterologous NAD⁺-dependent formate dehydrogenase (fdh) was synthesized to intracelluarly generate extra reducing power. The homologous thiolase (thl) was up-regulated to direct more flux from C2 to C4. A new mutant ACKKO-thl-fdh-adhE2 was constructed and characterized using free-cell fermentation in a 2-L bioreactor. It was found that the butanol titer was improved from 15 g/L to 19.5 g/L using the new mutant. The proteomics was used to further investigate how the cell engineering changed the expression of enzymes involved in the butanol formation. This study showed that the proteomics-guided rebalance of carbon and redox is an effective strategy to improve butanol production.