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 H2 fermentation, wild-type 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 H2 (193 and 284 nmol, respectively) than the wild type (66 nmol H2). 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 H2, whereas the ATP-enhanced strain (W3110+fhlA+pckA) produced 50% increased H2 (443 nmol). Total H2 in the NAD(P)H-enhanced strain (W3110+fhlA+maeA) was similar to that in the control strain at 319 nmol H2. 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.