A Strategy to Increase Microbial Hydrogen Production, Facilitating Intracellular Energy Reserves.

Overexpression of the genes encoding phosphoeneolpyruvate carboxykinase (pckA) and NAD-dependent malic enzyme (maeA) facilitates higher intracellular ATP and NAD(P)H concentrations, respectively, under aerobic conditions in Escherichia coli. To verify a hypothesis that higher intracellular energy reserves might contribute to H-2 fermentation, wildtype E. coli strains overexpressing pckA and maeA were cultured under anaerobic conditions in a glucose minimal medium. Overexpression of pckA and maeA enabled E. coli to produce 3-times and 4-times greater H-2 (193 and 284 nmol, respectively) than the wild type (66 nmol H-2). The pckA and maeA genes were further overexpressed in a hydrogenase-3-enhanced E. coli strain. The hydrogenase-3-enhanced strain (W3110+fhlA) produced 322 nmol H-2, whereas the ATP-enhanced strain (W3110+fhlA+pckA) produced 50% increased H-2 (443 nmol). Total H-2 in the NAD(P) H-enhanced strain (W3110+fhlA+maeA) was similar to that in the control strain at 319 nmol H-2. Possible explanations for the contribution of the increased cellular energy reserves to the enhanced hydrogen fermentation observed are discussed based on the viewpoint of metabolic engineering strategy.