Fermentative hydrogen production in recombinant Escherichia coli harboring a [FeFe]-hydrogenase gene isolated from Clostridium butyricum

The [FeFe]-hydrogenase (hydA) from Clostridium butyricum TERI BH05-2 strain was isolated to elucidate its molecular characterization. A 1953 bp DNA fragment encompassing the ORF and the putative promoter region of hydA gene was PCR amplified and subcloned into pGEM®-T-Easy cloning vector (pGEM®-T-hydA). The hydA DNA sequence revealed the presence of a 1725 bp length ORF (including the stop codon) encoding 574 amino acids with a predicted isoelectric point and molecular mass of 6.8 and 63097.67 Da, respectively. The hydA ORF was PCR amplified from pGEM®-T-hydA and inserted into a prokaryotic expression vector to create a recombinant plasmid (pGEX-5X-hydA) and transformed into Escherichia coli BL-21. The recombinant E. coli BL-21 was investigated for fermentative hydrogen production under anaerobic condition from glucose. Heterologous expression of the Clostridium butyricum hydA resulted in 1.9 fold increase in hydrogen productivity as compared to that from the wild type strain, C. butyricum TERI BH05-2. The hydrogen yield of the recombinant strain was 3.2 mol H2/mol glucose, 1.68 fold higher than the wild type parent strain.

► Fe–Fe hydrogenase catalyzes fermentative hydrogen production.
► Clostridium butyricum Fe–Fe hydrogenase gene was isolated & cloned in Escherichia coli BL-21.
► Hydrogenase gene sequence revealed the presence of a 1725 bp length ORF encoding 574 amino acids.
► Recombinant E. coli BL-21 demonstrated enhanced fermentative hydrogen production from glucose.
► Acetic acid was the major volatile fatty acid produced during hydrogen fermentation.