LAUSR

Abstract The combustion of fossil fuels is leading to global warming due to the emissions of greenhouse gases. Hydrogen (H2) is a clean and sustainable energy source and a promising alternative to fossil fuels. The bacteria Escherichia coli naturally produces H2, and it has been widely modified by metabolic engineering to enhance this production. Malate plays an important role in H2 synthesis when glycerol is used as a C source. In this work, C-flux was more efficiently redirected from malate to H2 by the PBAD promoter-controlled co-overexpression of the malate dehydrogenase and the malic enzyme MaeA in the dcuD and frdCdcuD mutants. These mutants previously have been reported as improved producing strains, but by using this strategy, H2 production increased 36 and 24%, respectively. The overexpression of both enzymes is essential, although MaeA expression was higher than Mdh, using the pBAD promoter, which was more appropriate than the pTrc99a one.