Biofuels that are renewable and environmentally benign constitute an important area of research, as the supply of fossil fuels decreases and the amount of green house gases in the atmosphere increases. Biohydrogen is not as well explored as other biofuels, but its properties render it a promising complement, as it is clean and can be used directly in fuel cells to generate electricity, the only waste products being water and heat. Hydrogenproducing microorganisms have the potential to be used to recycle industrial waste, such as carbohydrates from food manufacturing. Hence the cost of waste disposal could be reduced whilst biofuel is being produced through microbial processes.
Escherichia coli is a well-known microorganism that produces hydrogen under fermentative conditions, through the conversion of formate to hydrogen gas and carbon dioxide, via an enzyme complex called formate hydrogenlyase (FHL). The complex is anchored to the inner cell membrane and consists of seven subunits: a formate dehydrogenase, a [Ni-Fe] hydrogenase, three electron carrier proteins, which together make up a large ‘hydrophilic domain’, and two integral membrane proteins (the ‘membrane domain’).
Even though the entire bacterial genome is known, the FHL complex remains little understood and has proven difficult to isolate and characterise. During this project, a genetically modified strain producing only the hydrophilic domain of FHL was constructed, and the resultant sub-complex was purified. It was hoped that, if a stable and homogenous core complex could be isolated, it might be subjected to further analysis, such as elucidating the subunit stoichiometry and solving the structure.
Furthermore, FHL is notoriously oxygen labile, which hampers its study and technological development. However, oxygen tolerance is a natural feature found in some other [Ni-Fe] hydrogenases, and recent research shows that this property is likely dependent on the presence of extra cysteine residues near an important metal cluster in the enzyme. These cysteines are not present in FHL and a complex that could be active in both aerobic and anaerobic conditions may be a useful tool in optimising microbial biohydrogen processes. Thus, three strains that each expressed a modified FHL variant carrying single Cysteine-for-Glycine substitutions were constructed. The modified FHL complexes proved to remain active in vivo, and can serve as the basis of genetically engineering oxygen tolerance into this important enzyme.