Biologically-produced hydrogen (H2) or ?biohydrogen? is one promising source of renewable energy. A number of microorganisms are being studied as potential producers of biohydrogen through biophotolysis, indirect biophotolysis, photo-fermentations or dark-fermentations. Microorganisms produce H2 by the activity of either hydrogenases or nitrogenases: Hydrogenase enzymes catalyze the reaction: 2H+ + 2e- ? H2 whereas nitrogenases catalyze the reduction N2 with the following limiting stoichiometry: N2 + 8H+ + 8e- ? H2 + 2NH3. In this work, we have coordinated aspects of both pathways to develop optimized biocatalysts for hydrogen overproduction using the following steps: 1. Engineering a hydrogen responsive genetic circuit in the purple non-sulphur nitrogen-fixing bacterium Rhodobacter capsulatus SB1003: R. capsulatus carries nitrogenase and hydrogenase enzymes able to produce H2. It also carries a system to detect H2 that is composed of three proteins: a H2-sensor hydrogenase (HupUV), a histidine kinase (HupT) and a response regulator (NtrC-like transcription factor, HupR) (Vignais et al., 2005). In the presence of H2, this sensor triggers expression of hydrogenase structural and biosynthetic genes. Taking advantage of this system, we have introduced a reporter gene under the control of hupS promoter and removed the uptake hydrogenase, generating a new biological-sensor strain capable of accumulating and detecting the presence of both exogenous H2 and the H2 produced by its own nitrogenase. This biotechnological tool allows us to obtain a measurable and proportional signal when H2 is present in the cell. 2. Generating variants of the molybdenum nitrogenase structural genes nifH, nifD and nifK: we are using in vitro evolution techniques to perform random mutagenesis in these genes with a controlled mutation rate. The resulting variants were cloned under nifH promoter control into a broad-host-range vector (Kovach et al., 1995) optimized for diazotrophic conditions. Libraries obtained (around 4 x 106 clones) were introduced and expressed in the strain carrying the modified biological hydrogen sensor. The suitable combination of both tools results in the development of a genetic circuit for the high-throughput screening of H2 overproducing nitrogenase variants thus allowing detection and isolation of clones that present a significant signal increased, through the use of cell-sorting cytometry. Thus far, around 1500 clones have been successfully selected by this method, confirming the possibility of using the designed system to select hydrogen-overproducing enzymes.
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