Citrobacter amalonaticus Y19 (Y19) can produce hydrogen (H-2) from oxidation of carbon monoxide (CO) via the water-gas shift reaction. The reaction is catalyzed by two enzymes, carbon monoxide dehydrogenase (CODH) and carbon monoxide-dependent hydrogenase (CO-Hyd). The contig genome sequencing of Y19 exhibited the presence of unique CO oxidizing gene clusters encoding CODH (cooFS), CO-Hyd (cooMKLXUH) and a putative CO-responsive transcriptional activator (cooA). To improve CO-dependent H-2 production activity, we developed recombinant Y19 by homologously over-expressing cooA. The overexpression of cooA improved the whole-cell CO-dependent H-2 production activity (3.4-fold), and enzyme activities of CODH (5.3-fold) and CO-Hyd (1.2-fold). Furthermore, quantitative PCR analysis revealed a significant increase in the transcription of the genes located in CODH and CO-Hyd operons of recombinant Y19. The high CO-dependent H-2 production activity of the recombinant C. amalonaticus was stably maintained during repeated exposure to CO. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.