(408e) Engineering Enzymes for Hydrogen Production From Biomass

Hydrogen is a potential energy carrier to replace hydrocarbon fuels and reduce emissions from their consumption. Hydrogen production from renewable cellulosic biomass is favored for its nearly carbon-neutral cycle, placing high interest on its cost-effective technology development. We have previously demonstrated the feasibility of using an unpurified cell extract engineered for hydrogen production from glucose. The endogenous pentose phosphate pathway is coupled with a synthetic electron transfer pathway composed of three key proteins: ferredoxin-NADP⁺ reductase (FNR), ferredoxin, and a clostridial hydrogenase. However, the major challenge lies in increasing the electron flux from NADPH to hydrogen. In particular, low turnover numbers have been observed for the electron transfer from FNR to ferredoxin in the pathway. This work is aimed to investigate the application of FNR-hydrogenase fusion proteins for improved turnover number in hydrogen production. The fusion protein consists of FNR from Anabaena sp. and [Fe-Fe] hydrogenase from C. pasteurianum, physically linked by a polypeptide chain and expressed in E. coli strain. We also varied the linker length and its amino acid composition for increased electron transfer. Compared to un-fused enzymes added at equimolar concentrations, the fusion FNR-hydrogenase provides up to 5-fold improvement in hydrogen production rates.