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 (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.

Fig. 1

Gene organization of CO oxidation machinery (a) and carbon catabolite repression on expression of coo operon genes in C. amalonaticus Y19 (b). Binding of the transcriptional activator CooA in the intergenic regions of cooM and hypC is shown in (a). In b, the relative mRNA levels for various structural genes involved in CO-dependent H₂ production during cell growth on glucose and maltose, respectively, are shown. Cells were grown both in the presence and absence of CO

Fig. 2

Topology and sequence analysis of CO-regulated promoters in C. amalonaticus Y19. The putative CRP- and CooA-binding sites are indicated with arrows, and the promoter elements (−10/−35) are shown in boxes. The numbers in parentheses after CooA and/or CRP indicate matches between each inverted repeat and the consensus sequence motifs (CooA or CRP). The CooA-binding inverted repeat towards the CO-hyd operon is a perfectly symmetrical (9/9, 9/9), 9-bp match with that of the CooA/CRP operator reported in Rhodospirillum rubrum and other carboxydotrophs (TGTC(A/G)N6(C/T)GACA)

Fig. 3

Comparison of promoter strengths of various constitutively expressed genes in C. amalonaticus Y19. The relative mRNA abundance of the mutated version of narG* was estimated based on the mRNA abundance of narG in E. coli. C. amalonaticus Y19 was grown on glucose or maltose and in the presence of 20% CO

Fig. 4

Relative mRNA levels of genes involved in CO-dehydrogenase and CO-dependent hydrogenase in C. amalonaticus Y19 and promoter-replaced Y19-PR1 strain. The mRNA levels were compared with those of the reference gene, rpoD. C. amalonaticus Y19-PR1 recombinant was grown on glucose or maltose and in the presence and absence of 20% CO

Fig. 5

Time-course profiles of cell growth and H₂ production by wild-type Y19 and Y19-PR1 grown on maltose as carbon source. a, b Represent wild-type C. amalonaticus Y19 while c and d represent Y19-PR1. Each strain was grown in the absence (a, c) and presence (b, d) of CO. Symbols cell growth (open circle), CO consumption (closed circle), H₂ production (open square) and pH (cross)

Fig. 6

Time-course profiles of cell growth and H₂ production by wild-type Y19 and Y19-PR1 grown on glucose as carbon source. a, b Represent wild-type C. amalonaticus Y19 while c, d represent Y19-PR1. Each strain was grown in the absence (a, c) and presence (b, d) of CO. Symbols cell growth (open circle), CO consumption (closed circle), H₂ production (open squares) and pH (cross)