Empowering extra fuel supply in E. coli by electron bifurcation for robust H2, ATP and succinate production

Microbial production of hydrogen (future ideal fuel and important gas for industries) under anoxic conditions has limited ATP availability and low efficiency. We engineered E. coli K12 to acquire a flavin-based electron bifurcation (FBEB) system, a bioenergetic route typically found in strict anaerobes, which uses NADH to generate low potential reduced ferredoxin and high potential butyryl-CoA. The oxygen-tolerant FBEB-E. coli showed higher H2 and succinate production (2-4 folds), lower cellular reduction potentials, greater accumulation of cellular reductants and various metabolites, including ATP (up to a 7-fold increase). It could better tolerate prolonged and recycled usage of the engineered cell for H2 and succinate production than the native strain. FBEB-E. coli could also use various substrates such as formate, D-glucose and food waste for H2 and succinate production. This is a promising pathway to sustainable H2 and succinate production. This work also demonstrates that E. coli with an extra electron bifurcation system is a robust synthetic biology host.