Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli

• CODH of Citrobacter amalonaticus Y19 was heterologously expressed in Escherichia coli.
• No Y19 maturation proteins were needed for heterologous expression of this Ni–Fe–S metalloenzyme.
• The recombinant E. coli showed 3.5-fold increased CODH activity compared to C. amalonaticus Y19.
• Heterologously expressed CODH did not show CO-dependent H2 production in recombinant E. coli.

Carbon monoxide (CO) is highly toxic but is an abundant carbon source that can be utilized for the production of hydrogen (H2). CO-dependent H2 production is catalyzed by a unique enzyme complex composed of carbon monoxide dehydrogenase (CODH) and CO-dependent hydrogenase (CO–H2ase), both of which contain metal cluster(s). In this study, CODH and the required maturation proteins from the novel facultative anaerobic bacterium Citrobacter amalonaticus Y19 were cloned and heterologously expressed in Escherichia coli. For functional expression of CODH in E. coli, only CooF (ferredoxin-like protein) and CooS (CODH), not the maturation proteins, were needed. The recombinant E. coli BL21(DE3)-cooFS showed a 3.5-fold higher specific CODH activity (4.9 U mg protein−1) compared to C. amalonaticus Y19 (Y19) (1.4 U mg protein−1). Purified heterologous CODH from the soluble cell-free extract of the recombinant E. coli showed a specific activity of 170.6 U mg protein−1. Recombinant E. coli harboring Y19 CODH and maturation proteins did not produce H2 from CO, suggesting that the native hydrogenases present in E. coli could not substitute the Y19 CO–H2ase for CO-dependent H2 production.