Oxygen-dependent enhancement of hydrogen production by engineering bacterial hemoglobin in Escherichia coli

H₂ production under aerobic conditions has been proposed as an alternative method to overcome the fundamentally low yield of H₂ production by fermentative bacteria by maximizing the number of electrons that are available for H₂. Here, we engineered Vitreoscilla hemoglobin (VHb) in Escherichia coli to study the effects of this versatile oxygen (O₂)-binding protein on oxic H₂ production in a closed batch system that was supplemented with glucose. The H₂ yields that were obtained with the VHb-expressing E. coli were greatly enhanced in comparison to the negative control cells in culture that started with high O₂ tensions. The formate hydrogen lyase (FHL) activity of oxically cultured, VHb-expressing cells was also much higher than that of the negative control cells. Through inhibitor studies and time-course experiments, VHb was shown to contribute to the improved H₂ yield primarily by increasing the efficiency of cellular metabolism during the aerobic phase before the onset of H₂ production and not by working as an O₂-scavenger during H₂ production. This new approach allowed more substrate to remain to be further utilized for the production of more H₂ from limited resources. We expect that VHb can be successfully engineered in potential aerobic H₂-producing microbial systems to enhance the overall H₂ production yield. In addition, the remarkably high FHL activity of oxically grown, VHb-expressing cells may make this engineered strain an attractive whole-cell biocatalyst for converting formate to H₂.