Design and characterisation of synthetic operons for biohydrogen technology

Abstract

Biohydrogen is produced by a number of microbial systems and the commonly used host bacterium Escherichia coli naturally produces hydrogen under fermentation conditions. One approach to engineering additional hydrogen production pathways is to introduce non-native hydrogenases into E. coli. An attractive candidate is the soluble [NiFe]-hydrogenase from Ralstonia eutropha, which has been shown to link NADH/NAD+ biochemistry directly to hydrogen metabolism, an activity that E. coli does not perform. In this work, three synthetic operons were designed that code for the soluble hydrogenase and two different enzyme maturase systems. Interestingly, using this system, the recombinant soluble hydrogenase was found to be assembled by the native E. coli [NiFe]-hydrogenase assembly machinery, and, vice versa, the synthetic maturase operons were able to complement E. coli mutants defective in hydrogenase biosynthesis. The heterologously expressed soluble hydrogenase was found to be active and was shown to produce biohydrogen in vivo.

The structure of hydrogenase-2 from Escherichia coli: implications for H2-driven proton pumping

“…This observation suggested that overexpression of the hybO gene encoding the small subunit alone could be a useful strategy. To test this hypothesis, an E. coli mutant strain was chosen (IC011, Supplementary Table S1), which lacked the genes for the Hyd-1 and Hyd-3 large subunits and also carried in-frame, nonpolar deletions of hybO and hybA [ 20 ]. Although still encoding the Hyd-2 large subunit HybC, this strain has no hydrogenase activity.…”

“…Not only is the hyb operon repressed by IscR [ 15 ], but removal of the iscR gene also has proved beneficial for overproduction of Fe–S cluster-containing proteins [ 38 ]. A version of the IC011 strain carrying a Δ iscR allele (HJ001) [ 20 ] was transformed with pO N or pO C . Once more, the inclusion of an N-terminal His-tag appeared detrimental to protein stability and enzyme assembly, whereas the strain producing HybO with a C-terminal His-tag generated active Hyd-2 in excess of that observed for the native enzyme ( Figure 2A ).…”

“…To maximize [NiFe]-cofactor biosynthesis capability for HybC in the host strains, hyaB and hycE , which encode the large Ni- and Fe-containing subunits of Hyd-1 and Hyd-3, respectively, were deleted. It has previously been shown that a synthetic version of the R. eutropha hypA1B1F1C1D1E1X operon, encoding cofactor maturases, is active in E. coli [ 20 ]. A version of the hypA1-X operon, including the constitutive E. coli tat promoter [ 20 ], was integrated into the chromosome of strain HJ001 at the nonessential tatD locus to give the new strain ‘HJ001-hyp’ (see Figure 1B and Supplementary Table S1).…”

“…E. coli K-12 strains used in this work (Supplementary Table S1) are based on MC4100 [ 19 ]. IC011 (as MC4100, Δ hyaB , Δ hycE , Δ hybOA ) and HJ001 (as IC011 Δ iscR ) have been recently described [ 20 ]. The HJ001-hyp strain was prepared by first cloning a synthetic hypA1-X operon, based on the sequences of Ralstonia eutropha [NiFe]-hydrogenase maturases [ 20 ], into pFAT122 [ 21 ] as an EcoRI/SalI fragment.…”

“…IC011 (as MC4100, Δ hyaB , Δ hycE , Δ hybOA ) and HJ001 (as IC011 Δ iscR ) have been recently described [ 20 ]. The HJ001-hyp strain was prepared by first cloning a synthetic hypA1-X operon, based on the sequences of Ralstonia eutropha [NiFe]-hydrogenase maturases [ 20 ], into pFAT122 [ 21 ] as an EcoRI/SalI fragment. This strain was then combined with a SalI/KpnI fragment from pFAT123 [ 21 ], resulting in a pBluescript (Amp R ) vector carrying a Δ tatD :: hypA1-X allele.…”