(29s) Energy-Efficient Butanol Production By Clostridium Acetobutylicum with Histidine Kinase Regulation to Improve Strain Tolerance and Process Robustness

Clostridium acetobutylicum usually sporulates and halts its metabolism, which limits its use in industrial acetone–butanol–ethanol (ABE) fermentation. It is challenging to manipulate the highly regulated sporulation program used by clostridia for survival. Orphan histidine kinases (HKs), a global regulator, were found to control sporulation, cell growth, and metabolism. Single knockout of cac3319 aborted sporulation at the initiation stage but enhanced butanol tolerance and production, whereas cac0437 knockout caused premature sporulation and inhibited the transition from acidogenesis to solventogenesis, resulting in early autolysis and low butanol production. No solvent was produced when both cac3319 and cac0903 were knocked out. On the other hand, a mutant with double knockouts of cac3319 and cac0323 showed enhanced butanol production of >20 g/L by a repeated batch fermentation in a fibrous bed bioreactor, which enabled in situ product recovery via vapor stripping-vapor permeation to achieve the highest butanol production of >400 g/L ever reported. This study elucidated the regulatory roles of HKs in controlling clostridial sporulation and solventogenesis and demonstrated combinatory knockouts of HKs as a novel metabolic engineering approach to enhance butanol production suitable for industrial application.