Hydrogen production and metabolic flux analysis of metabolically engineered Escherichia coli strains

Escherichia coli can produce H2 from glucose via formate hydrogen lyase (FHL). In order to improve the H2 production rate and yield, metabolically engineered E. coli strains, which included pathway alterations in their H2 production and central carbon metabolism, were developed and characterized by batch experiments and metabolic flux analysis. Deletion of hycA, a negative regulator for FHL, resulted in twofold increase of FHL activity. Deletion of two uptake hydrogenases (1 (hya) and hydrogenase 2 (hyb)) increased H2 production yield from 1.20 mol/mol glucose to 1.48 mol/mol glucose. Deletion of lactate dehydrogenase (ldhA) and fumarate reductase (frdAB) further improved the H2 yield; 1.80 mol/mol glucose under high H2 pressure or 2.11 mol/mol glucose under reduced H2 pressure. Several batch experiments at varying concentrations of glucose (2.5–10 g/L) and yeast extract (0.3 or 3.0 g/L) were conducted for the strain containing all these genetic alternations, and their carbon and energy balances were analyzed. The metabolic flux analysis revealed that deletion of ldhA and frdAB directed most of the carbons from glucose to the glycolytic pathway leading to H2 production by FHL, not to the pentose phosphate pathway.