Expanding the substrates for a bacterial hydrogenlyase reaction

produces enzymes dedicated to hydrogen metabolism under anaerobic conditions. In particular, a formate hydrogenlyase (FHL) enzyme is responsible for the majority of hydrogen gas produced under fermentative conditions. FHL comprises a formate dehydrogenase (encoded by ) linked directly to [NiFe]-hydrogenase-3 (Hyd-3), and formate is the only natural substrate known for proton reduction by this hydrogenase. In this work, the possibility of engineering an alternative electron donor for hydrogen production has been explored. Rational design and genetic engineering led to the construction of a fusion between ferredoxin (Fd) and Hyd-3. The Fd-Hyd-3 fusion was found to evolve hydrogen when co-produced with pyruvate :: ferredoxin oxidoreductase (PFOR), which links pyruvate oxidation to the reduction of ferredoxin. Analysis of the key organic acids produced during fermentation suggested that the PFOR/Fd-Hyd-3 fusion system successfully diverted pyruvate onto a new pathway towards hydrogen production.