Enhanced butanol tolerance and production from puerariae slag hydrolysate by Clostridium beijerinckii through metabolic engineering and process regulation strategies.

Butanol is a more desirable second-generation biomass energy source. Acetone-butanol-ethanol (ABE) fermentation using Clostridium spp. is a promising method for butanol production. However, the toxicity of butanol to the producing strains leading to its low yield and the high cost of feedstock are the main obstacles limiting the ABE fermentation industry. In this study, to enhance the butanol tolerance and production in Clostridium beijerinckii D9, the strategies of metabolic engineering and process regulation were employed. With this effort, a recombinant strain D9/pykA was successfully developed. Furthermore, the effect of exogenous fermentation waste streams and their two-stage addition strategy on ABE fermentation was also investigated. Under the optimal condition, the highest butanol and total solvent production of 11.20 ± 0.58 g/L and 13.65 ± 0.51 g/L was achieved in C. beijerinckii D9/pykA, representing increases of 40.70 % and 37.05 %, respectively, compared to the original strain D9. Additionally, the results of the physiological mechanism revealed that the two-stage fermentation waste stream addition improved NADH synthesis and upregulated key genes involved in butanol biosynthesis, and thus enhancing the production. These insights could provide a foundation for further optimization of ABE fermentation processes and offer promising avenues for improving other similar research.