Construction and analysis of mutants of the hydrogen photoproduction machine in the model cyanobacterium Synechocystis

Photosynthetic organisms are attractive organisms for hydrogen production using water and solar energy, while preserving fresh water and arable soils without adding fertilizers. The model cyanobacterium Synechocystis PCC 6803 produces small and transitory amounts of H₂ thanks to its bidirectional [NiFe] hydrogenase Hox. The Hox complex with its 5 protein subunits (HoxEFUYH) catalyzes the reversible reaction 2H⁺ + 2e^- ↔ H₂. The [NiFe] catalytic site of the Hox enzyme is assembled using a six R subunits HypABCDEF complex and matured by the HoxW protease that cleaves HoxH and activates its [NiFe]-containing center. Engineering cyanobacteria for hydrogen production relies on a better understanding of the role of hydrogenase in the cyanobacterium metabolism. During my PhD, I have constructed and analyzed 7 sophisticated mutants of Synechocystis, allowing the simultaneous over-expression (constitutive or regulated by the growth temperature) of the hoxEFUYH and hypABCDEF genes. We demonstrated that the simultaneous over production of the HoxEFUYH and HypABCDEF proteins, combined to an increase in nickel availability led to an approximately 20 fold increase of the active hydrogenase level. Moreover, using a deleted hox operon mutant we showed that hydrogenase is dispensable in standard phototrophic growth conditions. Comparing the phenotypes of different mutants constructed in this study enables us to demonstrate for the first time that the hydrogenase operates in cell protection against oxidative stress (H₂O₂) and sugar stress (glucose or glycerol). Besides, I have also participated to the characterization of a new regulator (AbrB2) of the expression of the hydrogenase. This transcription factor represses the hoxEFUYH operon and is involved in the tolerance to stress induced by diamide or nickel. For the first time in cyanobacteria, are dox control of the activity of this regulator by a post-translational gluthathionylation was identified. Collectively, our findings showed that several genetic and physiological strategies should be combined in a single strain to strongly increase hydrogen production in Synechocystis. Meanwhile the presently constructed mutants proved to be very powerful tools to achieve this goal. (author)