Development of a synthetic pathway to convert glucose to hydrogen using cell free extracts

Sustainable production of biochemicals from cellulosic biomass is an attractive alternative to chemical production from fossil fuels. We describe the further development of a three protein synthetic pathway to convert NADPH and H+ to hydrogen using a ferredoxin-NADP+ reductase, a ferredoxin, and the [FeFe] hydrogenase from Clostridium pasteurianum at a rate greater than 14 mmol H2 L−1 hr−1 using natural enzymes. We also demonstrate the feasibility of coupling this pathway to a cell-free extract to convert glucose to hydrogen in a potentially cost effective manner. Both of these accomplishments serve as the basis for further engineering to optimize both the yield and productivity of a low-cost cell-free process for the production of highly reduced biochemicals.