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

Abstract

Background: Citrobacter amalonaticus Y19 is a good biocatalyst for production of hydrogen (H-2) from oxidation of carbon monoxide (CO) via the so-called water-gas-shift reaction (WGSR). It has a high H-2-production activity (23.83 mmol H-2 g(-1) cell h(-1)) from CO, and can grow well to a high density on various sugars. However, its H-2-production activity is expressed only when CO is present as an inducer and in the absence of glucose. Results: 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 similar to 10,000 colonies, showed a similar CO-dependent H-2-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. Conclusions: Convenient recombinant Y19-PR1 that expresses CO-dependent H-2-production activity without being limited by CO and glucose was obtained.