Electro-controlled distribution of reducing equivalents to boost isobutanol biosynthesis in microbial electro-fermentation of S. oneidensis

Efficient and directional supply of reducing equivalents is crucial for high-yield production of desired chemicals. Herein, an engineered isobutanol-producing Shewaenlla oneidensis was modularly constructed by assembling the electro-controlled distribution system of reducing equivalents and the isobutanol biosynthesis pathway. A dual-stage isobutanol electro-fermentation process was first established, including +0.5 V for cell growth and −0.6 V for isobutanol synthesis. Then, a redox biosensor-based dynamic regulation system was constructed to further decouple cell growth and isobutanol synthesis phases, enabling efficient supply of reducing equivalents. Lastly, an electro-controlled CRISPRi transcription inhibition system was designed to inhibit competitive metabolic pathways, which led to directional distribution of reducing equivalents and carbon flux toward isobutanol biosynthesis. Thus, the titer of isobutanol reached 1,321.5 ± 106.8 mg/L, a 10.8-fold increase from the original strain with 94.9% of the theoretical yield. This study achieved electro-controlled directional distribution of reducing equivalents and enhanced biosynthesis of reductive products via microbial electro-fermentation.