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 H from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H. As a result, their H 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 H production activities, but their sensitivity to O inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O. The diazotrophic filamentous cyanobacteria protect their nitrogenases from O 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 O amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 μmol-H 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 H production by an O-sensitive hydrogenase.
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.
Background: The ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen (H 2 ) is a promising alternative for renewable, clean-energy production. However, studies of the topic in the last decade have shown that much improvement is needed before sustainable cyanobacterial-based H 2 production becomes economically viable. In this study, we investigated the impact of inducing O 2 -consumption to enhance H 2 photoproduction yields in the heterocyst-forming, N 2 -fixing cyanobacterium Nostoc PCC7120. Results: The flv3B gene, encoding a flavodiiron protein naturally expressed in the heterocyst of Nostoc, was overexpressed. Compared to the wild type, the recombinant strain obtained displayed a significantly higher H 2 production under aerobic growth and phototrophic conditions. Nitrogenase activity assays indicated that flv3B overexpression did not increase the nitrogen fixation rates. On the other hand, quantitative RT-PCR experiments showed that the transcription of the hox genes, encoding the NiFe Hox hydrogenase was greatly elevated in the flv3B overexpressing strain. Conclusion: We conclude that the overproduced Flv3B protein might have enhanced O 2 -consumption, thus creating conditions inducing hox genes and facilitating H 2 production. The present study clearly demonstrates the potential to use metabolic engineered cyanobacteria for photosynthesis driven H 2 production.
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