Heterologous and homologous expression and characterization of a [Nife]-hydrogenase from the hyperthermophilic archaeon, Pyrococcus furiosus

Abstract

The reality that fossil fuels exist in finite amounts has shifted a major focus of research to renewable energy generation. Energy sources of the future must be abundant and carbon neutral with minimal impact on the environment. Hydrogen has the potential to satisfy these requirements as it is clean burning and has high energy content per mass unit, but current methods of production rely on non-renewable resources. The biological production from sunlight or biomass is an attractive alternative to current production methods. Hydrogenase, the biological catalyst responsible for activating gaseous hydrogen, has been the subject of research for more than 80 years. The long tenure this enzyme has enjoyed in the sciences is not a badge of honor; rather it is a testament to the extreme difficulty associated with researching this complex metalloenzyme. While previous efforts have elegantly elucidated the structure, catalytic mechanism, and factors involved in assembling this enzyme, it has not been possible to readily manipulate these proteins or generate large quantities for research purposes. The model chosen for these studies was Pyrococcus furiosus (Pf) a strictly anaerobic archaeaon that grows in shallow marine volcanic vents at temperatures near 100°C. These enzymes and pathways involved in hydrogen metabolism in Pf are being investigated by a combination of biochemical, bioinformatical, and molecular approaches. This research developed a novel method to heterologously express a soluble hydrogenase from Pf in E. coli yielding preparative amounts of thermostable, active enzyme. Following genetic advances in Pf a heterodimeric module of the normally heterotetrameric soluble hydrogenase was homologously overexpressed and produced a stable, active enzyme with altered coenzyme specificity.