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The biological production of hydrogen is an appealing approach to mitigating the environmental problems caused by the diminishing supply of fossil fuels and the need for greener energy. <i>Escherichia coli</i> is one of the best-characterized microorganisms capable of consuming glycerol—a waste product of the biodiesel industry—and producing H₂ and ethanol. However, the natural capacity of <i>E. coli</i> to generate these compounds is insufficient for commercial or industrial purposes. Metabolic engineering allows for the rewiring of the carbon source towards H₂ production, although the strategies for achieving this aim are difficult to foresee. In this work, we use metabolomics platforms through GC-MS and FT-IR techniques to detect metabolic bottlenecks in the engineered Δ<i>ldh</i>Δ<i>gnd</i>Δ<i>frdBC</i>::kan (M4) and Δ<i>ldh</i>Δ<i>gnd</i>Δ<i>frdBC</i>Δ<i>tdcE</i>::kan (M5) <i>E. coli</i> strains, previously reported as improved H₂ and ethanol producers. In the M5 strain, increased intracellular citrate and malate were detected by GC-MS. These metabolites can be redirected towards acetyl-CoA and formate by the overexpression of the citrate lyase (CIT) enzyme and by co-overexpressing the anaplerotic human phosphoenol pyruvate carboxykinase (hPEPCK) or malic (MaeA) enzymes using inducible promoter vectors. These strategies enhanced specific H₂ production by up to 1.25- and 1.49-fold, respectively, compared to the reference strains. Other parameters, such as ethanol and H₂ yields, were also enhanced. However, these vectors may provoke metabolic burden in anaerobic conditions. Therefore, alternative strategies for a tighter control of protein expression should be addressed in order to avoid undesirable effects in the metabolic network.