Design and characterisation of synthetic operons for biohydrogen technology

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.