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Most Cited
Improved Functional Expression of Carbon Monoxide Dehydrogenase from Carboxydothermus hydrogenoformans Using Genetically Engineered Escherichia coli Under Aerobic Conditions
Abstract
Carbon monoxide dehydrogenase derived from Carboxydothermus hydrogenoformans ( Ch CODH) is a representative anaerobic
enzyme that contains [4Fe–4S] clusters. To functionally express Ch CODH 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 Ch CODH alone under aerobic
conditions, a 10-fold and 2-fold increase in the specifi c 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 Ch CODH and the SUF system. Co-expression of Tig, a
chaperone protein, and the addition of 2 mM FeSO 4 , which constitutes the iron-sulfur cluster, further increased the total
activity up to 97,758 U/L. Moreover, the amount of Fe incorporated into Ch CODH was proportional to the specifi c activity,
while the amount of Ni was not correlated with the specifi c 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.
