Cyanobacterial Oxygen-tolerant Hydrogenases: functional characterization and engineering – OtolHyd

Hydrogenases (H2ases) catalyze the reversible conversion of molecular hydrogen to protons and electrons. They have attracted considerable interest since H2 is considered as an environmentally friendly energy carrier. Coupling H2 bio-production to photosynthesis has emerged as a promising approach and the use of photosynthetic organisms such as cyanobacteria has been tested worldwide. Unfortunately, the H2ases of cyanobacteria turned out to be sensitive to O2 inhibition which hinders their use in conjunction with photosynthesis. The [NiFe] H2ases have been classified as either standard or O2-tolerant based on their ability to function in the presence of O2. These enzymes have evolved structural and catalytic adaptations which make of them promising biotechnologically tools. Recently, a phylum wide genomic survey of H2ase genes in cyanobacterial genomes has allowed us to identify for the first time the presence of genes encoding O2-tolerant H2ases in this phylum. This finding opens new perspectives for the use of cyanobacteria in H2 evolution. Our project falls within this frame work since it aims at characterizing the function and the role of O2-tolerant H2ase in two cyanobacterial strains: Cyanothece PCC 7425 and Leptolyngbya boryana PCC 6306. The expression of the O2-tolerant H2ase encoding genes in these organisms will be characterized. Mutant deleted of these genes will be obtained and their phenotypes studied. The enzymes will be purified and their catalytic activity in the presence or absence of O2 measured. We will also explore the possibility of using these enzymes to couple H2 production to photosynthesis. In this context, if the enzymes will be found to oxidize H2, site directed mutagenesis will be employed to rewire their catalytic activity towards H2 evolution. Finally, the engineered enzyme and the proteins required to its maturation will be produced in the fast growing cyanobacterium Synechococcus elongatus PCC 7942.