A recombinant plasmid pETD-SL was constructed to analyze the effect of hydrogen production by [NiFe] hydrogenase gene isolated from Rhodopeseudomonas palustris. A two-promoter vector pETDuet-1 was used to construct pETD-SL which contains hupS and hupL gene, and then pETD-SL was transferred into recombinant Escherichia coli BL21(DE3) which was named E. coli BH20. Furthermore, HupS and HupL protein in E. coli BH20 were detected by SDS-PAGE analysis. Finally, the hydrogen production from E. coli BH20 was determined in the hydrogen production complex medium containing 4.40 g glucose. The results showed E. coli BH20 evolves 0.32 +/- 0.01 mol hydrogen/mol glucose compared with the wild type non-hydrogen-production strain E. coli BL21(DE3) under anaerobic condition. However, we found the recombinant protein has a high activity which altered the normal metabolism of E. coli BL20. Furthermore, this study demonstrated non-native [NiFe] hydrogenase has potential for metabolic engineering to enhance hydrogen yields in E. coli. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.