Overproduction of the Flv3B flavodiiron, enhances the photobiological hydrogen production by the nitrogen-fixing cyanobacterium Nostoc PCC 7120.

BACKGROUND: The ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen (H2) is a promising alternative for renewable, clean-energy production. However, the most recent, related studies point out that much improvement is needed for sustainable cyanobacterial-based H2 production to become economically viable. In this study, we investigated the impact of induced O2-consumption on H2 photoproduction yields in the heterocyte-forming, N2-fixing cyanobacterium Nostoc PCC7120. RESULTS: The flv3B gene, encoding a flavodiiron protein naturally expressed in Nostoc heterocytes, was overexpressed. Under aerobic and phototrophic growth conditions, the recombinant strain displayed a significantly higher H2 production than the wild type. Nitrogenase activity assays indicated that flv3B overexpression did not enhance the nitrogen fixation rates. Interestingly, the transcription of the hox genes, encoding the NiFe Hox hydrogenase, was significantly elevated, as shown by the quantitative RT-PCR analyses. CONCLUSION: We conclude that the overproduced Flv3B protein might have enhanced O2-consumption, thus creating conditions inducing hox genes and facilitating H2 production. The present study clearly demonstrates the potential to use metabolic engineered cyanobacteria for photosynthesis driven H2 production.

The Pkn22 Kinase of Nostoc PCC 7120 Is Required for Cell Differentiation via the Phosphorylation of HetR on a Residue Highly Conserved in Genomes of Heterocyst-Forming Cyanobacteria.

Hanks-type kinases encoding genes are present in most cyanobacterial genomes. Despite their widespread pattern of conservation, little is known so far about their role because their substrates and the conditions triggering their activation are poorly known. Here we report that under diazotrophic conditions, normal heterocyst differentiation and growth of the filamentous cyanobacterium Nostoc PCC 7120 require the presence of the Pkn22 kinase, which is induced under combined nitrogen starvation conditions. By analyzing the phenotype of pkn22 mutant overexpressing genes belonging to the regulatory cascade initiating the development program, an epistatic relationship was found to exist between this kinase and the master regulator of differentiation, HetR. The results obtained using a bacterial two hybrid approach indicated that Pkn22 and HetR interact, and the use of a genetic screen inducing the loss of this interaction showed that residues of HetR which are essential for this interaction to occur are also crucial to HetR activity both in vitro and in vivo. Mass spectrometry showed that HetR co-produced with the Pkn22 kinase in Escherichia coli is phosphorylated on Serine 130 residue. Phosphoablative substitution of this residue impaired the ability of the strain to undergo cell differentiation, while its phosphomimetic substitution increased the number of heterocysts formed. The Serine 130 residue is part of a highly conserved sequence in filamentous cyanobacterial strains differentiating heterocysts. Heterologous complementation assays showed that the presence of this domain is necessary for heterocyst induction. We propose that the phosphorylation of HetR might have been acquired to control heterocyst differentiation.

Phototrophic hydrogen production from a clostridial [FeFe] hydrogenase expressed in the heterocysts of the cyanobacterium Nostoc PCC 7120.

The conversion of solar energy into hydrogen represents a highly attractive strategy for the production of renewable energies. Photosynthetic microorganisms have the ability to produce H2 from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H2 production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H2. As a result, their H2 production rates are low, which makes them not suitable for a high yield production. On the other hand, [FeFe] enzymes originating from anaerobic organisms such as Clostridium exhibit much higher H2 production activities, but their sensitivity to O2 inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O2. The diazotrophic filamentous cyanobacteria protect their nitrogenases from O2 by differentiating micro-oxic cells called heterocysts. Producing [FeFe] hydrogenase in the heterocyst is an attractive strategy to take advantage of their potential in a photosynthetic microorganism. Here, we present a biological engineering approach for producing an active [FeFe] hydrogenase (HydA) from Clostridium acetobutylicum in the heterocysts of the filamentous cyanobacterium Nostoc PCC7120. To further decrease the O2 amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 µmol-H2 per mg Chlorophyll a, which represents 20-fold the amount produced by the wild type strain. This result is a clear demonstration that it is possible to associate oxygenic photosynthesis with H2 production by an O2-sensitive hydrogenase.