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Abstract
In the prospect of engineering cyanobacteria for the biological photoproduction of hydrogen, we have studied the hydrogen production machine in the model unicellular strain Synechocystis PCC6803 through gene deletion, and overexpression (constitutive or controlled by the growth temperature). We demonstrate that the hydrogenase-encoding hoxEFUYH operon is dispensable to standard photoautotrophic growth in absence of stress, and it operates in cell defense against oxidative (H2O2) and sugar (glucose and glycerol) stresses. Furthermore, we showed that the simultaneous over-production of the proteins HoxEFUYH and HypABCDE (assembly of hydrogenase), combined to an increase in nickel availability, led to an approximately 20-fold increase in the level of active hydrogenase. These novel results and mutants have major implications for those interested in hydrogenase, hydrogen production and redox metabolism, and their connections with environmental conditions.
Highlights
Cyanobacteria are the only known prokaryotes capable of oxygenic photosynthesis, which uses nature’s most abundant resources, solar energy, water, CO2 and mineral nutrients, to produce a large part of the oxygen and organic assimilates for the aerobic food chain
The absence of the hoxEFUYH operon in the DhoxEFUYH::Kmr mutant was confirmed upon the analysis of culture grown for a few generations in absence of Km to stop counter-selecting the propagation of possibly remaining wild-type (WT) chromosome copies, prior to the PCR assays (Figure S4)
We confirmed through quantitative RT-PCR that the DhoxEFUYH::Kmr mutant completely lacks hoxEFUYH transcripts, as well as the HoxF and HoxH proteins and hydrogenase activity (Figure S5). These data, together with the fact that the DhoxEFUYH::Kmr mutant grows as healthy as the wild-type strain (WT) strain under standard photoautotrophic conditions (Figure S5), showed that the hoxEFUYH operon is dispensable in Synechocystis
Summary
Cyanobacteria are the only known prokaryotes capable of oxygenic photosynthesis, which uses nature’s most abundant resources, solar energy, water, CO2 and mineral nutrients, to produce a large part of the oxygen and organic assimilates for the aerobic food chain. The pentameric hydrogenase enzyme (HoxEFUYH; Hox for hydrogen oxidation), which is reversibly inactivated by oxygen [7], is a bidirectional enzyme with a bias to H2 production [8]. This reaction, 2H+ + 2e2 « H2, uses NAD(P)H as the source of electrons originating from photosynthesis and/or sugar catabolism, and a nickel-iron center and several iron-sulfur clusters as redox cofactors [9]. The Hox complex is assembled by the six-subunits HypABCDEF complex enzyme [2,9] encoded by the hypABCDEF genes, which are scattered onto the chromosome (Figure S1). Physiological studies indicated that the hydrogenase enzyme acts as an emergency electron valve to release excess of photosynthetic electrons, for instance during the transition from (anaerobic) dark to light conditions, leading to weak and transient H2 production [7,9]
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