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Biotechnol Biofuels. 2017 Mar 28;10:80. doi: 10.1186/s13068-017-0770-8. eCollection 2017.

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

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1
School of Chemical and Biomolecular Engineering, Pusan National University, San 30, Jangeon-dong, Geumjeong-gu, Busan, 609-735 Republic of Korea.

Abstract

BACKGROUND:

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.

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 ~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.

CONCLUSIONS:

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

KEYWORDS:

CO-Hyd; CODH; Citrobacter amalonaticus; Water–gas-shift reaction; gapA; narG

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