Improved Functional Expression of Carbon Monoxide Dehydrogenase from Carboxydothermus hydrogenoformans Using Genetically Engineered Escherichia coli Under Aerobic Conditions

Carbon monoxide dehydrogenase derived from Carboxydothermus hydrogenoformans (ChCODH) is a representative anaerobic enzyme that contains [4Fe–4S] clusters. To functionally express ChCODH under aerobic conditions in genetically engineered E. coli, co-expression with the SUF system that facilitates the assembly of iron–sulfur clusters under oxidative stress and iron limitation was performed. Results showed that compared to the expression of ChCODH alone under aerobic conditions, a 10-fold and 2-fold increase in the specific activity and protein yield, respectively, resulting in a total activity of up to 29,185 U/L was observed in E. coli co-expressed with ChCODH and the SUF system. Co-expression of Tig, a chaperone protein, and the addition of 2 mM FeSO₄, which constitutes the iron-sulfur cluster, further increased the total activity up to 97,758 U/L. Moreover, the amount of Fe incorporated into ChCODH was proportional to the specific activity, while the amount of Ni was not correlated with the specific activity. Finally, high cell density cultivation under optimized aerobic conditions in the SUF system and the chaperone protein Tig co-expressed in recombinant E. coli resulted in a total activity of up to 235,689 U/L.