Improvement of Hydrogen Production Yield by Rebalancing NADPH/NADH Ratio in a Recombinant Escherichia coli

The NAD(P)H- or ferredoxin-dependent hydrogen production pathway is known to have a huge potential for improving its production yield compared to the formate hydrogen lyase (FHL) route. To improve the hydrogen production yield through this pathway, rebalancing the NADPH/NADH ratio based on the cofactor preference is important, in addition to enhancing the NAD(P)H pool by reducing the competing pathways. NADPH:ferredoxin oxidoreductase coupled with [FeFe]-hydrogenase preferably utilizes NADPH as an electron donor. Therefore, directing NADPH should be required to improve the hydrogen production yield. For balancing the NADPH/NADH ratio, nicotinamide nucleotide transhydrogenase has a key role, and Escherichia coli has the two transhydrogenases, PntAB and SthA. In this study, several recombinants were constructed to up-regulate pntAB or to down-regulate sthA in the competing-ldhA-deleted E. coli expressing [FeFe]-hydrogenase system. As a result, an about 3.9-fold increased hydrogen yield was observed in the pntAB-overexpressed and sthA-deleted strain. Therefore, the combination of ldhA down-regulation and NADPH/NADH ratio rebalancing via transhydrogenase engineering can improve the hydrogen production yield in the NADPH- or ferredoxin-dependent hydrogen production pathway.