Discovery and characterisation of new enzymes for hydrogen production and green chemistry

Using hydrogen as a sustainable fuel and energy carrier requires tackling several technical challenges, including the implementation of clean and efficient production technologies from renewable resources. H2 is produced in nature by several microorganisms that rely on hydrogenases, a vast group of complex metallo-enzymes that catalyse the key reaction in H2 metabolism.
Between the three phylogenetically distinct classes of hydrogenases, [FeFe]-hydrogenases are crucial in physiological H2 production. Consequently, they received extensive attention for possible biotechnological applications. For example, [FeFe]-hydrogenases have been engineered and overexpressed in suitable cellular chassis for both dark and light-driven H2 production from waste biomass or water, respectively. Moreover, purified [FeFe]-hydrogenases have been immobilised on semiconductor nanoparticles and utilised to direct storage of solar energy into H2.
One of the main factors limiting a broader exploitation of [FeFe]-hydrogenases is their sensitivity to atmospheric oxygen: O2 is a powerful inhibitor and inhibition is usually irreversible, but some examples of O2-stable [FeFe]-hydrogenases have recently been reported.

Identifying novel [FeFe]-hydrogenases with improved performances (for example, faster turnover rates and/or higher stability) will be crucial to exploit them in industrial processes. Currently, practical exploitation is hindered by the fact that only a small number of these enzymes have been experimentally characterised in detail, and this limits the "catalogue" of enzymes available for any given application.
The core aims of this PhD project are: 1) to provide a more comprehensive view of the diversity of [FeFe]-hydrogenases; 2) to significantly expand the toolbox of available enzymes to be exploited for H2 production technologies; 3) to explore any unconventional reactivity of [FeFe]-hydrogenases to be exploited in green chemistry processes.

In the initial phase of the project, the student will use bioinformatics tools to identify novel [FeFe]-hydrogenases in silico, by performing sequence studies and building structural models. This phase will aim at identifying genes with unconventional features that could positively influence the enzyme performances, such as the presence of additional enzyme subunits or specific residues that may allegedly influence catalysis.
Subsequently, building on the expertise acquired during the rotation project, the student will move to the wet lab and produce a selection of the best targets (via cloning of the selected genes, recombinant overexpression and purification). Established methods to incorporate the catalytic cluster in vivo (via the native/heterologous HydEFG maturation system) or in vitro (via the pre-made synthetic cofactor 2FeMIM) will be used.
The new enzymes will be characterised for their specific activity (reaction rate for H2 production and H2 oxidation) and for their stability (particularly towards temperature, solvents and O2). Biophysical characterisation will also be performed including spectroscopy, crystallography, and X-ray diffraction in collaboration with an external partner, Dr Stephen Carr (Research Complex at Harwell).
The new enzyme(s) will also be tested for the ability to reduce/oxidise useful cofactors, such as NAD(P)+/NAD(P)H, or unnatural substrates that can highlight new horizons in the industrial exploitation of these enzymes within green chemistry processes. Protein engineering approaches will be considered to improve the performances and/or to redirect the reactivity towards artificial substrates.