Citrobacter amalonaticus Y19 for constitutive expression of carbon monoxide-dependent hydrogen-production machinery

Citrobacter amalonaticus Y19 is a good biocatalyst for production of hydrogen (H₂) from oxidation of carbon monoxide (CO) via the so-called water-gas-shift reaction (WGSR). It has a high H₂-production activity (23.83 mmol H₂ g^-1 cell h^-1) from CO, and can grow well to a high density on various sugars. However, its H₂-production activity is expressed only when CO is present as an inducer and in the absence of glucose.

In order to avoid dependency on CO and glucose, in the present study, the native CO-inducible promoters of WGSR operons (CO dehydrogenase, CODH, and CODH-dependent hydrogenase, CO-hyd) in Y19 were carefully analyzed and replaced with strong and constitutive promoters screened from Y19. One engineered strain (Y19-PR1), selected from three positive ones after screening ~10,000 colonies, showed a similar CO-dependent H₂-production activity to that of wild-type Y19, without being affected by glucose and/or CO. Compared with wild-type Y19, transcription of the CODH operon in Y19-PR1 increased 1.5-fold, although that of the CO-hyd operon remained at a similar level. To enhance the activity of CO-Hyd in Y19-PR1, further modifications, including an increase in gene copy number and engineering of the 5' untranslated region, were attempted, but without success.

Convenient recombinant Y19-PR1 that expresses CO-dependent H₂-production activity without being limited by CO and glucose was obtained.