(176y) Modulating NAD+/NADH Via Redox-Sensing Transcriptional Repressor Rex Knockout for Enhanced n-Butanol Production in Clostridium Tyrobutyricum

Biobutanol is a superior biofuel to ethanol. Recently, the engineered strain of Clostridium tyrobutyricum overexpressing aldehyde/alcohol dehydrogenase encoded by adhE2 has garnered significant attention as a prime candidate for n-butanol production from renewable biomass feedstock. However, the availability of reducing equivalents, primarily NADH, is crucial for butanol biosynthesis. In this study, the redox-sensing transcriptional repressor Rex in C. tyrobutyricum was knocked out via CRSPR-Cas genome editing and replaced with adhE2 to channel more NADH towards alcohol biosynthesis. The mutant ΔRex::adhE2 produced 15.4 g/L butanol in serum bottle fermentation with the addition of 50 μM methyl viologen (MV). Metabolic flux analysis and qRT-PCR results showed that Rex knockout promoted butanol biosynthesis and C4 metabolism in the fermentation. However, a significant level of lactate was also produced, underscoring the importance of further enhancing C4 metabolic flux. Subsequently, overexpressing an exogenous NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase (HBD) in this mutant further improved butanol production to 17.8 g/L, with a high butanol yield of 0.33 g/g glucose. A high volumetric productivity of 0.42 g/L/h was obtained in fed-batch fermentation carried out in a 3-L bioreactor at 37 ^oC. The present study elucidated the roles of Rex in modulating NADH and NADPH-dependent HBD in enhancing C4 metabolic flux, which underscored their significance as effective strategies for enhancing butanol biosynthesis in C. tyrobutyricum.