Engineering Clostridium Tyrobutyricum for High Butanol Production through Induction Expression of Exogenous...

Clostridium tyrobutyricum Δcat1::adhE2 is a promising cell factory for butanol production because of its robustness, high butanol tolerance, and minimal butyrate production. However, excessive acetate and ethanol production remains a major bottleneck limiting its butanol yield. Coexpressing an exogenous hbd(Ck) encoding the NADPH-dependent 3-hydroxybutyryl-CoA dehydrogenase (HBD) from Clostridium kluyveri with adhE2 could increase the C4 carbon flux, resulting in increased butanol and decreased acetate and ethanol production. However, constitutively overexpressing hbd(Ck) in Δcat1::adhE2 shows little improvement in butanol yield, productivity, and selectivity, which might be caused by redox imbalance and growth inhibition. To alleviate this problem, C. tyrobutyricum MΔcat1::adhE2-Pbgal-hbd(Ck) with a dynamic expression of hbd(Ck) controlled by an inducible promoter was developed. In serum bottle fermentation at 37 °C, when the hbd(Ck) expression was induced at 12 h or in the early exponential phase, butanol production increased ∼20% in yield (from 0.22 to 0.27 g/g glucose), 87.5% in productivity (from 0.16 to 0.30 g/L·h), and 52% in selectivity (from 0.46 to 0.70 g/g total products) compared to the control strain without expressing any hbd(Ck), whereas hbd(Ck) expression induced at 0 or 24 h in MΔcat1::adhE2-Pbgal-hbd(Ck) or constitutively in MΔcat1::adhE2-Pcat1-hbd(Ck) showed significantly lower butanol yield and productivity. At 25 °C, MΔcat1::adhE2-Pbgal-hbd(Ck) with 12 h induction produced the highest butanol titer of 23 g/L with 0.32 g/g yield, 0.16 g/L·h productivity, and 0.83 g/g product selectivity due to much reduced acetate formation. Subsequent scale-up to a stirred-tank bioreactor at 37 °C increased productivity to 0.39 g/L·h while also achieving high butanol titer (21.8 g/L), yield (0.30 g/g), and selectivity (0.67 g/g). The optimized induction timing resulted in a balanced NAD(P)H pool, effectively channeling substrates toward butanol biosynthesis. It was concluded that the timing for hbd(Ck) expression was critical as it affected glucose catabolism, cell growth, redox balance, and carbon flux distribution. These findings underscore the potential of dynamic metabolic regulation to overcome bottlenecks in biobutanol production, providing a scalable and economically viable bioprocess for industrial application.