Metabolic Engineering of C. Tyrobutyricum for High n-Butanol Production By Rebalancing Carbon and Redox (Rapid Fire)

Biobutanol is a sustainable and environmentally friendly fuel that could be used as the substitute of gasoline. The acidogenic Clostridium tyrobutyricum had been engineered, ACKKO-adhE2, to produce butanol. However, our previous Omics study showed that the redox was not balanced and some enzymes involved in carbon metabolism had low expression in ACKKO-adhE2. In this study, both carbon engineering and redox engineering were performed to achieve high butanol production. The heterologous NAD⁺-dependent formate dehydrogenase (fdh) was expressed in ACKKO-adhE2 to synthesize a NADH producing pathway. The thiolase (thl) that directs metabolic flux from acetyl-CoA to acetocetyl-CoA was also overexpressed. Two mutant strains, ACKKO-adhE2-fdh and ACKKO-thl-adhE2-fdh, were constructed. The free-cell fermentation showed that butanol titer was increased to 19.03 g/L by ACKKO-adhE2-fdh. The ACKKO-thl-adhE2-fdh further improved butanol production, selectivity and yield. To obtain a stable strain, an advanced single plasmid was constructed by optimizing the promoter and terminator. This study demonstrated that the carbon and redox rebalance in C. tyrobutyricum enabled a high production of butanol.