Deletion of iscR stimulates recombinant clostridial Fe-Fe hydrogenase activity and H₂-accumulation in Escherichia coli BL21(DE3)

Proteins that catalyze H₂-pathways often contain iron-sulfur (Fe-S) clusters and are sensitive to O₂. We tested whether deletion of the gene encoding the transcriptional negative regulator, IscR, could enhance the ability of Escherichia coli BL21 to synthesize active recombinant H₂-pathway components and stimulate ferredoxin-dependent H₂-accumulation in the presence or absence of oxygen. Under anoxic conditions, deletion of iscR stimulated recombinant Fe-Fe hydrogenase activity threefold, whilst plasmid-based overexpression of the isc operon had no effect on hydrogenase activity. After cultivation with 21% (v/v) O₂ in the headspace, no recombinant hydrogenase activity was observed in soluble extracts of wild-type BL21, although low levels of activity could be observed in the ΔiscR strain (700-fold lower than anoxic conditions, 180-fold greater than the limit of detection). Under closed batch conditions starting with 5% (v/v) O₂, ΔiscR strains displayed fivefold greater levels of total hydrogenase activity and recombinant ferredoxin-dependent H₂-accumulation relative to the control strain. In cultures starting with 10% (v/v) O₂, H₂-accumulation was stimulated 35-fold relative to the control. ΔiscR strains displayed enhanced synthesis and activity of integral H₂-pathway components under all tested conditions and enhanced H₂-accumulation under partially oxic conditions. Deletion of iscR is, therefore, a useful strategy to stimulate H₂-production, particularly if the hydrogenase catalyzes the rate-limiting reaction.