Clostridium tyrobutyricum, a high-butyrate producer, was engineered to produce butanol by overexpressing aldehyde/alcohol dehydrogenase (adhE2). However, butanol biosynthesis by the engineered strains was limited by the NADH availability. In this study, we first knocked out the transcriptional repressor Rex, a key sensor of the NADH/NAD⁺ ratio, in the genome of C. tyrobutyricum with adhE2 insertion, using the endogenous CRISPR/Cas genome-editing system. The ΔRex::adhE2 mutant strain (ΔR) showed increased butanol and ethanol production, resulting from upregulated bcs operon and improved NADH availability through metabolic rewiring. To reinforce the reducing equivalent supply and carbon flux toward butanol biosynthesis, an NAD⁺/NADH kinase (NADK) and NADPH-dependent β-hydroxybutyryl-CoA dehydrogenase (HBD) were introduced into the ΔR strain. The resulting mutant strain ΔR-NH produced butanol at an increased yield of 0.25 g/g glucose. We further optimized carbon reallocation through regulating cell division by expressing MinC, a key cell division inhibitor, in C. tyrobutyricum ΔR-NH, which increased the carbon recovery ratio by 11.48% and butanol yield to 0.30 g/g in the mutant ΔR-NMH. Butanol yield further increased to 0.31 g/g when methyl viologen (MV) was present in the fermentation medium. This study presents a novel metabolic engineering strategy for enhancing biobutanol production by synergistically manipulating metabolic flux, redox balance, and cell division control in C. tyrobutyricum.
Keywords: C. tyrobutyricum, Rex, NADH, Cell division, Carbon reallocation, Butanol