Photosynthetic microbes can be utilized for hydrogen production, generating a clean, carbon neutral energy carrier from abundant substrates. Cyanobacteria are photosynthetic prokaryotes with large potential for biotechnological energy applications and several strains are capable of hydrogen production. This production is catalysed by a bi-directional [NiFe]-hydrogenase, or by nitrogenase during nitrogen fixation. However, nature’s foremost hydrogen producing enzymes, the [FeFe]-hydrogenases, are not present in these organisms. Many [FeFe]-hydrogenases boast incredible catalytic activities and high bias towards proton reduction. Introduction of a suitable [FeFe]-hydrogenase in a cyanobacterial host could greatly improve the hydrogen production capacity. Unfortunately, generation and characterisation of cyanobacterial strains carrying active [FeFe]-hydrogenases is stalled by the intricate maturation process associated with these enzymes.

In this thesis, I investigate heterologous expression and artificial maturation of [FeFe]-hydrogenases in cyanobacteria. Genetic tools to reliably express [FeFe]-hydrogenases were developed and tested in the unicellular cyanobacterium Synechocystis PCC 6083, and in heterocysts of the filamentous cyanobacterium Nostoc punctiforme ATCC 29133. Following heterologous expression, functional, semisynthetic [FeFe]-hydrognases operating in vivo in cyanobacterial cells were generated by synthetic activation. The procedure proved successful in both the unicellular and filamentous strain, and for [FeFe]-hydrogenases from different groups and subclasses. The semisynthetic enzymes proved capable of hydrogen production under different environmental conditions and links to the metabolism of the host cell. Hydrogen production capacity proved long-lived and was retained for several days. In Nostoc punctiforme, synthetic activation was confirmed to generate active [FeFe]-hydrogenase in both vegetative cells and heterocyst.

The results presented in this thesis demonstrate a novel way to explore in vivo hydrogen production from heterologous [FeFe]-hydrogenases in cyanobacteria. In the search for suitable candidates for H₂ production systems, synthetic activation may be used to investigate a wide range of [FeFe]-hydrogenases, strains and cultivation conditions, circumventing the need of elaborate maturation machinery optimisation.