Construction of a synthetic YdbK-dependent pyruvate:H₂ pathway in Escherichia coli BL21(DE3)

A synthetic pyruvate:H₂ pathway was constructed in Escherichia coli BL21(DE3) by co-expression of six proteins: E. coli YdbK, Clostridium pasteurianum [4Fe-4S]-ferredoxin, and Clostridium acetobutylicum HydF, HydE, HydG, and HydA. The effect of cofactor addition and host strain on H₂ yield and fermentation product accumulation was studied, together with in vitro reconstitution of the entire pathway. The deletion of iscR and/or the addition of thiamine pyrophosphate to the medium enhanced the total and specific activity of recombinant YdbK and increased the yield of H₂ per glucose. It was concluded that the introduced pathway outcompeted other pyruvate-consuming reactions, and that the ability to compete for pyruvate at least in part was determined by total YdbK activity. The results demonstrate the successful construction of a high-yielding H₂ pathway in a microorganism that effectively does not synthesize any H₂. The additional co-expression of Bacillus subtilis AmyE enabled starch-dependent H₂ synthesis in minimal media.